Exploiting oxygen inhibited photopolymerization within emulsion droplets for the fabrication of microparticles with customizable properties

ABSTRACT

Described are methods and devices for the generation of hydrogel particles with micrometer and submicrometer dimensions using oxygen-inhibited partial polymerization, and the particles generated therefrom. The described methods generate particles with dimensions independent of the starting polymerizable solution dimension, for example, a microdroplet. Further, microfluidic flow parameters (e.g. viscosity, flow rate) and photopolymerization process parameters (e.g. optical exposure intensity and duration) are controlled to generate particles with tunable crosslinking density-determined properties including elasticity, diffusivity, and biomolecular display for diverse applications such as drug delivery, tissue engineering cell scaffolds, and single- and multiple- cell therapeutics. Similarly, gradients of crosslinking density-determined properties can be created within single particles through the selection of optical exposure intensity and duration. In addition to conventional spherical shapes, a suite of non-spherical shapes may be generated by manipulating the dimensions of the microfluidic channels and other related physical and process parameters.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. § 371 of International Application No. PCT/US2018/056237, filedOct. 17, 2018, which application claims the benefit of U.S. ProvisionalApplication Ser. No. 62/573,576 filed Oct. 17, 2017 and 62/586,680 filedNov. 15, 2017, each of which is hereby incorporated by reference in itsentirety, to the extent not inconsistent herewith.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under 1254608 awarded bythe National Science Foundation (NSF) and under P20 GM103432 awarded bythe National Institutes of Health (NIH). The government has certainrights in the invention.”

BACKGROUND OF INVENTION

PEG-based hydrogels have become widely used as drug delivery and tissuescaffolding materials. Common among PEG hydrogel-forming polymers arephotopolymerizable acrylates in the form of polyethylene glycoldiacrylate (PEGDA). Microfluidics and microfabrication technologies haverecently enabled the miniaturization of PEGDA structures, thus enablingmany possible applications for nano- and micro- structured hydrogels.The presence of oxygen, however, inhibits the photopolymerization ofPEGDA, which in turn frustrates hydrogel formation in environments ofpersistently high oxygen concentration. By developing an integratedmodel incorporating photoinitiation, reaction kinetics and oxygendiffusion, it is possible to utilize diffused oxygen to partiallypolymerize microdroplets, allowing for controlled generation ofmicroparticles smaller than the microdroplet undergoing polymerization.

Photopolymerization also plays an important role in numerous industrialand research applications, including biomaterials for cell encapsulationand delivery. A common design of hydrogel materials for cellencapsulation is the use of diacrylated macromers. The presence ofoxygen is known to inhibit the photopolymerization of PEGDA, but doesnot require mitigation on the macroscale reactions, and most notably, itlimits polymerization carried out in air permeable polydimethylsiloxane(PDMS) microfluidic devices. As an example, we present innovativemicrofluidic devices using PDMS along with silicate or glass surfaces(which prevent the diffusion of oxygen) to further control the partialpolymerization of PEGDA microdroplets.

Inverse colloidal crystals (ICCs) are the product of a lost waxfabrication method in which colloidal particles are assembled intoordered matrices in the presence of a liquid continuous phase. Followingsolidification of the continuous phase, particles are subsequentlyextracted, leaving behind a structured pore network. ICCs have beendeveloped for a variety of scientific and technological applications,yet their utility remains limited by harsh processing conditionsrequired to solubilize particles for pore framework formation. In thisexample, we present a new approach to ICC construction based uponphotodegradable polyethylene glycol diacrylate particle synthesis.Because the degradation of particulate phase requires only opticalillumination, particle assemblies can be eroded within tightly confiningmicrochannels, creating microfluidic-integrated ICCs. Using thisapproach, photodegradable particle assemblies are used to pattern porouspolyethylene glycol hydrogel network structure and interconnectivity.The non-invasive, gentle erosion of photodegradable PEG or PEGDAparticles allows secondary objects to be embedded within the pores ofthe ICC. While the presence of oxygen may be harmful to biologicalparticles present in the formation of the microparticles, precisecontrol of the oxygen solubility and diffusivity may be used to generatean oxygen gradient in the microdroplet during polymerization, allowingfor lower concentrations of oxygen in the polymerized microparticle.This approach is also facile, gentle and cytocompatible, indicating thatit holds great potential for structuring functional biomaterials.

SUMMARY OF THE INVENTION

Described herein are methods and devices for the generation of hydrogelparticles with micrometer and submicrometer dimensions usingoxygen-inhibited partial polymerization, and the particles generatedtherefrom. The described methods are versatile, and may generateparticles with dimensions independent of the starting polymerizablesolution dimension, for example, a microdroplet. Further, microfluidicflow parameters (e.g. viscosity, flow rate) and photopolymerizationprocess parameters (e.g. optical exposure intensity and duration) may becontrolled to generate particles with tunable crosslinkingdensity-determined properties including elasticity, diffusivity, andbiomolecular display for diverse applications such as drug delivery,tissue engineering cell scaffolds, and single- and multiple- celltherapeutics. Similarly, gradients of crosslinking density-determinedproperties can be created within single particles through the selectionof optical exposure intensity and duration. In addition to conventionalspherical shapes, a suite of non-spherical shapes may be generated bymanipulating the dimensions of the microfluidic channels and otherrelated physical and process parameters.

The described methods and devices may control polymerization bymanipulating various polymerization parameters such as initiatorconcentration, monomer or macromer concentration, oxygen concentration,oxygen diffusivity and oxygen solubility. All parameters may becontrolled in both the microdroplet and the surrounding non-aqueousphase. In the case of photopolymerization using ultraviolet (UV) light,the intensity of the light and exposure time may also be varied forcontrolled polymerization, allowing for unique and variablemicroparticle properties including degree of cross-linking, size,surface elasticity and biocompatibility.

In an aspect, provided is a method of generating a plurality ofmicroparticles comprising: a) providing a continuous phase comprising anon-aqueous liquid and a dispersed phase comprising an aqueous solutionhaving a monomer or a macromer and a photoinitiator; b) forming acomposition comprising microdroplets of the aqueous phase and thenon-aqueous phase, wherein oxygen is diffused through the non-aqueousphase into the microdroplets; and c) partially polymerizing the aqueousphase thereby generating a microparticle within the aqueous phase havinga smaller primary cross-sectional dimension than the microdroplet. In anembodiment, for example, the monomer or macromer comprises PEGDA and/orthe photoinitiator is Irgacure 2959, LAP or a combination thereof. Theaqueous phase may comprise 1% to 75%, 10% to 75%, or optionally, 10% to50% monomer or macromer by weight. In embodiments, the aqueous phasecomprises less than 10%, 0.1% to 10%, or optionally, 0.1% to 5%photoinitiator by weight.

The primary of the microparticle may be independent of the diameter ofthe microdroplet. The diffusion of oxygen into the microdroplet maygenerate an oxygen concentration gradient in the aqueous phase. Afterpartial polymerization, the oxygen concentration gradient may result ina crosslinking gradient in the plurality of microparticles. Continuouscrosslinking chain integration may be formed along the crosslinkinggradient. The step of partially polymerizing the aqueous phase may beoxygen inhibited. The aqueous phase may comprise 1% to 99%, 1% to 50%,10% to 50%, 20% to 75% or optionally, 20% to 50% monomer or macromer byweight. The aqueous phase may comprise 0.01% to 99%, 0.01% to 25%, 0.01%to 10%, or optionally, 0.01% to 5% photoinitiator by weight.

The non-aqueous phase may comprise a fluorocarbon oil or a hydrocarbonoil. The aqueous phase may have an oxygen concentration selected fromthe range of 0.1 mol/m³ to 2 mol/m³, 0.1 mol/m³ to 1 mol/m³, 0.5 mol/m³to 1 mol/m³, 0.5 mol/m³ to 2 mol/m³, or optionally, 0.1 mol/m³ to 0.5mol/m³. The non-aqueous phase may have an oxygen concentration selectedfrom the range of 2 mol/m³ to 5 mol/m³, 2 mol/m³ to 10 mol/m³, 2 mol/m³to 3 mol/m³, 1 mol/m³ to 5 mol/m³, or optionally, 1 mol/m³ to 3 mol/m³.

The aqueous phase may have an oxygen diffusivity selected from the rangeof 0.0001 mm²/s to 0.01 mm²/s, 0.001 mm²/s to 0.01 mm²/s, 0.001 mm²/s to0.1 mm²/s, or optionally, 0.0001 mm²/s to 0.001 mm²/s. The non-aqueousphase may have an oxygen diffusivity selected from the range of 0.0001mm²/s to 0.05 mm²/s, 0.0001 mm²/s to 0.1 mm²/s, 0.001 mm²/s to 0.01mm²/s, or optionally, 0.001 mm²/s to 0.05 mm²/s.

The step of partially polymerizing the aqueous phase may be carried outby exposure to UV light. The exposure of UV light may be carried out for1 ms to 1500 ms, 10 ms to 1000 ms, 1 ms to 1000 ms, or optionally 50 msto 550 ms. The exposure to UV light may be carried out for less than 750ms, 550 ms, 400 ms, or optionally, less than 250 ms.

The UV light may be provided at an intensity selected from the range of0.01 mW/cm² to 3 mW/cm², 0.01 mW/cm² to 2 mW/cm², 0.1 mW/cm² to 3mW/cm², or optionally, 0.1 mW/cm² to 2 mW/cm².

The aqueous phase and the non-aqueous phase may be formed in amicrofluidic device. The described methods may further comprise flowingthe microdroplets through one or more channels of the microfluidicdevice. The one or more channels may have a cross-sectional area lessthan or equal to 10000 μm², 5000 μm², 4000 μm², 2500 μm², or optionally,1000 μm². Flowing the microdroplets through the one or more channels maygenerate non-uniform microdroplets, for example, non-sphericalmicrodroplets.

The microfluidic device may comprise PDMS, glass or any combinationthereof. The PDMS may have an oxygen concentration selected from therange of 4 mol/m³ to 6 mol/m³, 3 mol/m³ to 5 mol/m³, 4.5 mol/m³ to 5.5mol/m³, or optionally, 4.5 mol/m³ to 5 mol/m³. The PDMS may have anoxygen diffusivity selected from the range of 0.01 mm²/s to 0.05 mm²/s,0.001 mm²/s to 0.1 mm²/s, 0.01 mm²/s to 0.1 mm²/s, or optionally, 0.001mm²/s to 0.05 mm²/s.

The microfluidic particle may have a primary cross-sectional dimensionof less than or equal to 30 μm, 20 μm, 10 μm, or optionally, 5 μm. Themicrodroplets may have an average primary cross-sectional dimension ofless than or equal to 200 μm, 100 μm, 75 μm, or optionally, 50 μm. Themicrodroplets may be substantially spherical. The microdroplets may beoblong, a disk, a biconcave disk, a torus, a rod, a wire, a bullet, acaterpillar or a horseshoe.

The microparticle may be bioactive. A surface of the microparticle maybe treated with a biological material, for example, biotin. A surface ofthe microparticle may have increased bioactivity. The aqueous phase mayfurther comprise a biological material.

In an aspect, provided is microparticle generated by the methodsdescribed herein.

In an aspect, provided is a microparticle comprising PEGDA having aprimary cross-sectional dimension of less than or equal to 20 microns.In embodiments, for example, the microparticle is non-spherical and hasa shape selected from the group comprising: oblong, a disk, a biconcavedisk, a torus, a rod, a wire, a bullet, a caterpillar and a horseshoe.

In an aspect, provided is a method of preparing a composite hydrogelwith comprising the steps of: a) providing a continuous phase comprisinga non-aqueous liquid and a dispersed phase comprising an aqueoussolution having a photodegradable monomer or a photodegradable macromerand a photoinitiator; b) forming a composition comprising microdropletsof the aqueous phase and the non-aqueous phase, wherein oxygen isdiffused through the non-aqueous phase into the microdroplets; c)partially polymerizing the aqueous phase thereby generating aphotodegradable microparticle within each of the microdroplets, each ofthe photodegradable microparticles having a smaller primarycross-sectional dimension than the microdroplet; d) removing excessaqueous phase from the photodegradable microparticles; e) at leastpartially encapsulating the photodegradable microparticles within anon-photodegradable polymer; and f) photodegrading the photodegradablemicroparticles to produce a composite porous hydrogel. The describedmethods may further comprise contacting the composite porous hydrogelwith a biological material. The composite hydrogel may have pore shapesin any of the non-spherical microparticle shapes described herein.

The described method may further comprise a step of generating an oxygenconcentration gradient in the aqueous phase. After partialpolymerization, the oxygen concentration gradient may result in acrosslinking gradient along a physical dimension of the inversehydrogel. Continuous crosslinking chain integration may be formed alongthe crosslinking gradient

In an aspect, provided is a method for preparing an inverse colloidalcrystal containing biological material comprising the steps of: a)providing a continuous phase comprising a non-aqueous liquid and adispersed phase comprising an aqueous solution comprising aphotodegradable monomer or a photodegradable macromer, the biologicalmaterial and an initiator; b) forming a composition comprisingmicrodroplets of the aqueous phase and the non-aqueous phase, whereinoxygen is diffused through the non-aqueous phase into the microdroplets;c) purging the composition comprising the microdroplets and thenon-aqueous liquid with an oxygen-free gas; d) partially polymerizingthe aqueous phase thereby generating a photodegradable microparticlewithin each of the microdroplets, each of the photodegradablemicroparticles having a smaller primary cross-sectional dimension thanthe microdroplet; e) removing excess aqueous phase from thephotodegradable microparticles; f) at least partially encapsulating thephotodegradable microparticles within a non-photodegradable polymer; andg) photodegrading the photodegradable microparticles to produce aninverse colloidal crystal having a plurality of pores containing abiological material. The inverse colloidal crystal may have pore shapesin any of the non-spherical microparticle shapes described herein

The described method may further comprise a step of generating an oxygenconcentration gradient in the aqueous phase. After partialpolymerization, the oxygen concentration gradient may result in acrosslinking gradient along a physical dimension of the inversecolloidal crystal. Continuous crosslinking chain integration may beformed along the crosslinking gradient. Greater than or equal to 80%,90%, or optionally 95% of the biological material may viable afterperforming the method.

Without wishing to be bound by any particular theory, there may bediscussion herein of beliefs or understandings of underlying principlesrelating to the devices and methods disclosed herein. It is recognizedthat regardless of the ultimate correctness of any mechanisticexplanation or hypothesis, an embodiment of the invention cannonetheless be operative and useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . provides an overview or graphical abstract of oxygen-inhibitedphotopolymerization.

FIG. 2A. Schematic of droplet formation and continuousphotopolymerization of oxygen-inhibited PEGDA. FIG. 2B.Photopolymerization proceeds within the droplet in an oxygen-dependentmanner, starting at the droplet center and ceasing where the steadystate oxygen concentration rises above the inhibition threshold. FIG.2C. Imaged droplets after photopolymerization reveal a sharp contrast atthe hydrogel particle boundary.

FIG. 3A. Plot of hydrogel particle radius as a function of exposure timeand intensity. Dashed horizontal lines indicate particle radii at agiven intensity after 30 s of UV exposure. Particles appear following aninduction time, indicated by vertical dotted lines, after which theirsize remains constant. ([PEGDA 700]=0.5M, [LAP]=17 mM) FIG. 3B. Imagesof a droplet being photopolymerized in a microfluidic channel atdifferent exposure times. Longer exposure times result in a visibleincrease in crosslinking, while the particle diameter (blue lines)remains constant. ([PEGDA 700]=0.3 M, [LAP]=17 mM, I=1.77 mW/cm²)

FIG. 4A. Emulsion droplets within fluorocarbon oils maintain a constantinterfacial oxygen concentration during photopolymerization. Blue dotsrepresent individual data points for the unpolymerized shell thicknessin droplets at a given distance from the nearest aqueous droplet. Dottedred line indicates unpolymerized shell thickness in droplets polymerizedunder flow conditions (u=990 μm/s, Re=0.177). ([PEGDA 700]=0.5 M,[LAP]=17 mM, I=1.77 mW/cm²) FIG. 4B. Experiments to assess kineticparameters and their effect upon steady state hydrogel particle sizewere conducted in quiescent reservoirs, which match continuous flowconditions. ([PEGDA 700]=0.5 M, [LAP]=17 mM).

FIG. 5 . Acrylate functional group concentration contributes to theregulation of unpolymerized shell thickness. Decreasing PEGDAconcentration produces a clear trend toward larger thicker unpolymerizedshell, which highlights its ability to vary the rate of oxygenconsumption and, in turn, the diameter of photopolymerized particles.([LAP]=17 mM).

FIGS. 6A-6D. PEG architecture and acrylate concentration regulateunpolymerized shell thickness. FIG. 6A. For constant acrylateconcentration, unpolymerized shell thickness varies considerably whenchanging PEGDA molecular weight from 700 to 575. FIG. 6B. Removing theinhibitor (MEHQ) from PEGDA 575 does not affect particlephotopolymerization. FIG. 6C. Adding PEGMA while varying PEGDAconcentration in order to maintain constant acrylate concentrationyielded no change in unpolymerized shell thickness. FIG. 6D. UnreactivePEG400 changed particle size by modifying solution viscosity. ([LAP]=17mM).

FIG. 7 . Photoinitiator (LAP) concentration and intensity regulatereaction kinetics and oxygen consumption, providing broad control overshell thickness. ([PEGDA 700]=0.5 M).

FIG. 8 . A comparison of two common photoinitiators (LAP vs. Irgacure2959) reveal stark kinetic differences, which correspond to largevariations in shell thickness over significant differences in initiatorconcentration. While these differences are dramatic, they are not nearlyas extreme as predicted by Equation 2 in conjunction with literaturevalues for extinction coefficient and quantum yield. An empiricaldetermination of an initiator-dependent rate constant instead allows thedroplet-size independent shell thickness, and therefore producedparticle size, to be predicted for a given hydrogel forming solutioncomposition. ([PEGDA 700]=0.5 M).

FIGS. 9A-9D. Predicted unpolymerized shell thicknesses under differentoperating conditions closely resemble empirical data. FIGS. 9A-9B. Themodel requires an empirically fitted diffusivity value to obtain a goodprediction when varying PEGDA concentration and molecular weight,signaling the importance of molecular transport in the photopolymerizedsystem. FIGS. 9C-9D. Model predictions closely match empirical data whenchanging initiator concentration, while kinetic differences between thetwo initiators were not as significant as previously reported.

FIG. 10 . provides an example devices for droplet microfluidics.

FIG. 11 . illustrates Oxygen Inhibited Droplet Photopolymerization.

FIG. 12 . demonstrates that particle size may be controlled.

FIG. 13 . provides examples of oblong and disk shaped particles.

FIG. 14 . provides examples of rod and wire shaped particles.

FIG. 15 . provides examples of bullet shaped particles as well asparticles that have been received multiple exposures.

FIG. 16 . illustrates that surfactant may be removed from the surface bynot fully polymerizing the particle.

FIG. 17 . illustrates that Acrylate-PEG-Biotin can be cross-linked intothe hydrogel matrix.

FIG. 18 . shows fluorescent intensity correlates to biotin groupspresent at the interface. Particles exposed for less time show increasedsurface roughness.

FIG. 19 . Surface activity decreases with increasing exposure time dueto crosslinking gradient at the particle surface.

FIGS. 20A-20B. Schematic of effect of interfacial bonding on hydrogelproperties. FIG. 20A. Particles with gradient crosslinking density atsurface leads to good interfacial bonding compared to particles withuniform and complete surface-crosslinking; FIG. 20B. composite with goodinterface shows stiffness similar to bulk hydrogel and significantlyhigher strength compared to composite with poor interface. FIG. 20Bshows compression testing result of bulk and composite hydrogel samplesmade from PEG30 hydrogel.

FIGS. 21A-B. FIG. 21A. Bulk hydrogel sample by bonding two parts at a45° flat internal surface. The internal surface was fully polymerized ifpolymerized against glass or partially polymerized if polymerizedagainst PDMS. FIG. 21B. Stress vs. Strain plots for bulk samples areshown beside samples made from two parts bonded at 45° surface. Thebonded samples had bulk material similar elastic modulus but low failurestrength and the PEG30 sample bonded at surface polymerized againstglass was extremely weak.

FIG. 22 . (Left) Schematic diagram of the experimental setup to observethe hydrogel network at the fully- and partially-polymerized hydrogelsurface. (Right) Fluorescent images show that gradual increase of thefluorescent at the interface for hydrogel polymerized in Air compared tosteep increase for hydrogel polymerized in Nitrogen (This result isexpected, however cannot be confirmed from the current results. Need toimage in higher magnification). rhodamine at the surface polymerizedagainst PDMS.

FIG. 23A. Composite hydrogel with hydrogel particles fromvortex-suspension and oil-column method as well as hydrogel pieces.Composite hydrogel with vortex particles shows lower elastic moduluswhile composite with particles from oil-column shows bulk materialsimilar elastic modulus. FIG. 23B. Composite made with soft-matrix andstiff-particles from oil-column method shows reinforcement, butparticles from vortex suspension method deteriorate the overallcomposite behavior. Composites with small pieces from bulk hydrogel asdiscontinuous phase showed bulk hydrogel-similar modulus, but lowerstrength compared to composite with oil-column particles.

FIG. 24A. Schematic of cell encapsulation in the particles fromoil-column method; FIG. 24B. Fluorescent image shows very high cellviability (˜90%) in the particles made by oil-column method. The greenand red dots indicate live and dead cells respectively; FIG. 24C.Composite hydrogels made from cell-laden particles; FIG. 24D.Fluorescent image shows live and dead cells inside the particlessurrounded by matrix material; FIG. 24E. Zoomed in fluorescent image ofparticles inside shows high cell viability is retained through thematrix polymerization step to fabricate composite hydrogel; FIG. 24F. Ascomparison to the composite, bulk matrix hydrogels shows very low cellviability.

FIGS. 25A-25D. Composite with various volume fractions of vortexparticles.

FIG. 26 . Various composites with vortex particles match cartilagebehavior showing possible applications.

FIG. 27 . Strain controlled test of composites with high volume fractionof stiff-vortex-particles shows debonding.

FIG. 28 . Cleaning of vortex-particles with ethanol and acetone did notimprove the composite modulus. This confirms that presence of surfactantin vortex particles cannot cause the poor interfacial bonding.

FIGS. 29A-29C. Size-controlled hydrogel particles with different shapes,such as oblong shapes (FIG. 29A), disks (FIG. 29B), and rods (FIG. 29C),can be produced in a high throughput fashion using droplet microfluidicsin combination with oxygen-inhibited particle photopolymerization.Aqueous and oil phase flow conditions determine droplet volume; channelgeometry dictates particle shape and aspect ratio; the particles finaldimensions can be tuned using oxygen-inhibited photopolymerization.

FIG. 30 . Droplet deformation when using heavy mineral oil(viscosity>130 cP) combined with in situ photopolymerization providesthe means to produce uniquely shaped particles from transient shapes.(Channel dimensions: width=100 μm; height=90 μm).

FIG. 31 . Multiple exposure particles can possess regionally distinctmechanical properties.

FIG. 32 . Using glass-bonded PDMS microfluidic devices can producehydrogel particles with crosslinking gradients due to asymmetric oxygendiffusivity profiles. In the pictures, elongated droplets werepolymerized into wires with crosslinking gradients, resulting in diversecoiling patterns. (25 wt % PEGDA 700, 0.5% LAP).

FIG. 33 . Schematic of surface-functionalized hydrogel particlefabrication via oxygen-inhibited photopolymerization of droplets withina microfluidic device.

FIGS. 34A-34B. Influence of photopolymerization exposure time (30% PEGDA700, 0.5% LAP, 1.0% Ac-PEG-Biotin 2 k): (FIG. 34A) Quantified florescentIntensity, and (FIG. 34B) images of availability of functional sites onhydrogel particle surface. Top row and bottom row are images of the sameparticles under different light filters, with each column having thesame exposure time (0.223 s, 0.294 s and 0.441 s, respectively).

FIG. 35 . Measured oxygen solubility (left axis) and relative oxygensolubility (right axis) in PEGDA solutions. Oxygen solubility isinversely proportional to PEGDA concentration; a significant differencein oxygen solubility between PEGDA 700 and PEGDA 575 solutions at equalacrylate concentrations was not observed.

FIG. 36 . Measured viscosity of PEGDA solutions (left axis) and inverseof the model fitted oxygen diffusivity coefficient (right axis) show aclose correlation between these two variables. This justifies the use offitted oxygen diffusivity in the reaction-diffusion model andestablishes a need to incorporate viscosity data into this model toobtain accurate predictions.

FIGS. 37A-37D. Sensitivity analysis for oxygen solubility anddiffusivity in the developed reaction-diffusion model for a droplet withradius=50 μm. Oxygen concentration profile (FIGS. 37A-37B) and extent ofmonomer conversion (FIGS. 37C-37D) along droplet radius show littlesensitivity to oxygen solubility, with very slight variation in theoxygen concentration profile and no change in predicted shell thickness.FIGS. 37B and 37D represent zoomed in selection for oxygen concentrationprofile and extent of monomer conversion respectively, marked in FIGS.37A and 37C as dotted squares. Model is highly sensitive to variationsin oxygen diffusivity, resulting from changes in solution viscosity.Black dotted line in D represents the cut off extent of reaction underwhich no gelation is observed (Model parameters: k_(d)=0.005 s⁻¹, [M]=1M, [PI]=17.0 mM LAP).

FIG. 38 . Measured fluorescent light source spectral output at 100%intensity (blue) and theoretical molar extinction coefficient for LAPand Irgacure 2959 (green).¹ The area under the overlap of these twocurves is used to predict k_(d) values for each initiator (Equation S2).LAP has significantly higher activity in the wavelength range used, butempirical results coupled with a fitted model have determined thedifference in the activity of these two initiators is not as substantialas the theoretical data predicts.

FIGS. 39A-B. Oxygen-inhibited photopolymerization of droplets containingAcryl-PEG-biotin produces functional hydrogels with surfactant-freesurfaces. FIG. 39A Schematic illustration of hydrogel microparticlefabrication via oxygen-inhibited photopolymerization. FIG. 39BMicrographs illustrating the process described in FIG. 39A, with NA-Rhfluorescence imaging showing the presence of a radial crosslinkingdensity gradient in the hydrogel network. Droplet content: 30 wt % PEGDA700, 0.5 wt % LAP, 0.5 wt % Acryl-PEG-Biotin, exposed for 700 ms, 350ρW/cm².

FIG. 40 . A Biotin/Neutravidin-Rhodamine (NA-Rh) assay reveals thatNeutravidin (NA) penetration is limited to a radial distance that isdictated by photopolymerization conditions. Elucidating the governingreaction-diffusion behavior allows the network architecture to bedefined, dictating local hydrogel mechanical properties and biomoleculardiffusion for applications such as drug release or bioassays. r_(max):radial position of the maximum florescence intensity.

FIG. 41 . Experimental fluorescent intensity (green) andreaction-diffusion model prediction of extent of conversion (blue)illustrate that the penetration of fluorescently tagged Neutravidin (NA)into the hydrogel particle is the consequence of constrained networkarchitecture at increasing conversion. Above a threshold extent ofconversion, the hydrogel mesh can no longer accommodate the diffusion ofNeutravidin. 38% represents this threshold conversion value, while 2% isthe extent required to achieve gelation.

FIG. 42 . Calculated NA penetration depth (dotted line), as determinedby an extent of conversion threshold value of 38%, accurately predictempirical observations.

FIG. 43 . illustrates the interfacial benefit provided by generating acrosslink gradient by utilizing oxygen inhibition during crosslinking.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

As used herein, the term “polymer” refers to a molecule composed ofrepeating structural units connected by covalent chemical bonds oftencharacterized by a substantial number of repeating units (e.g., equal toor greater than 3 repeating units, optionally, in some embodiments equalto or greater than 10 repeating units, in some embodiments greater orequal to 30 repeating units) and a high molecular weight (e.g. greaterthan or equal to 20,000 Da, in some embodiments greater than or equal to50,000 Da or greater than or equal to 100,000 Da). Polymers are commonlythe polymerization product of one or more monomer or macromerprecursors. The term polymer includes homopolymers, or polymersconsisting essentially of a single repeating monomer subunit. The termpolymer also includes copolymers which are formed when two or moredifferent types of monomers are linked in the same polymer. Usefulpolymers include organic polymers or inorganic polymers that may be inamorphous, semi-amorphous, crystalline or semi-crystalline states.

“Monomer” and/or “Macromer” each refer to a reagent which can undergopolymerization under appropriate conditions. A monomer or macromerreagent comprises at least one monomer or macromer molecule, where amonomer or macromer molecule is a molecule which can undergopolymerization, thereby contributing constitutional units to thestructure of a polymer or oligomer. In an embodiment, a monomer ormacromer reagent may be represented by an average or dominant chemicalstructure and comprise monomer molecules having that chemical structurebut may also contain components with other chemical structures. Forexample, a monomer or macromer reagent may comprise impurities havingchemical structures other than the average or dominant structure of thereagent. Macromer may refer to a reagent which is polymeric, e.g., has anumber of repeating units but may further undergo polymerization to forma polymer of macromer repeating units. In embodiments, for example,macromer refers to reagents having a high molecular weight (e.g. greaterthan or equal to 200 Da, in some embodiments greater than or equal to1000 Da or greater than or equal to 10,000 Da).

“Non-photodegradable polymer” refers to a polymer is that is notphotodegradable under selected exposure conditions, e.g., selectedwavelength range, intensity, power or a combination thereof. In anembodiment, for example, a non-photodegradable polymer refers to apolymer that does not degrade under the conditions present to degradephotodegradable polymers as described herein.

“Microparticles” refers to particles including polymers, havingrelatively small dimensions including diameter, radius, height, width,depth, etc. In embodiments, for example, microparticles refer toparticles having a lateral dimension (e.g. diameter) of less than orequal to equal to 1 mm. In some embodiments, microparticles refers toparticles having an average or mean diameter of less than or equal to100 μm, less than or equal to 50 μm, or less than or equal to 20 μm. Insome embodiments, microparticles are microspheres. In some embodiments,microparticles refer to particles having lateral dimensions selectedfrom the range of 10 nm to 1000 μm, preferably for some embodiments, 10nm to 100 μm.

“Microdroplets” refer to microparticles in the liquid phase. Asdescribed herein, microdroplet dimensions may be larger than thecorresponding generated microparticle, as only a portion of themicrodroplet is polymerized. For example, in some embodiments,microdroplets refer to droplets having a mean or average diameter ofless than or equal to 500 μm, less than or equal to 100 μm, or less thanor equal to 50 μm. In embodiments, microdroplets refer to liquids in asuspension, for example an emulsion. In an embodiment, microdropletsrefer to aqueous liquids suspended in a non-aqueous liquid. In someembodiments, microdroplets refer to particles having lateral dimensionsselected from the range of 10 nm to 1000 μm, preferably for someembodiments, 10 nm to 100 μm.

“Hydrogel” refers to an at least partially hydrophilic substance havingcharacterized by high water absorbency. In embodiments, hydrogelcomprises an at least partially hydrophilic polymer, superabsorbentpolymer or biomacromolecule, for example in a network configuration.Hydrogels may be characterized as a water swollen but insolublesubstance. In embodiments, for example, hydrogels may absorb watergreater than or equal to 10 times the hydrogel weight, greater than orequal to 50 times the hydrogel weight or, optionally, greater than orequal to 100 times the hydrogel weight.

“Primary cross-sectional dimension” refers to the largestcross-sectional dimension of a particle as described herein. Forexample, for a sphere the primary cross-sectional dimension is adiameter, while for a cylinder or a wire the primary cross-sectionaldimension is the diameter of the cross-sectional circle or ellipse atthe widest point along the axial length. Similarly, primarycross-sectional dimension may refer to the effective diameter of thecross-section of the described shape or particle.

Example 1—Precise Control of PEGDA Hydrogel Particle Size by OxygenInhibited Photopolymerization within Microfluidic Droplets Abstract

Hydrogels based on poly(ethylene glycol) (PEGDA) have been engineeredfor a variety of biomedical applications including drug delivery, celldelivery, and tissue engineering. The miniaturization of these materialsto nanoscale and microscale particles has been a subject of intenseactivity, and promises to extend their range of applicability. Ingeneral, however, these efforts have been frustrated by the inhibitionof chain growth polymerization by oxygen, an effect that is exacerbatedas target length scales are reduced. Here, we report a method thatexploits the undesirable oxygen-inhibited photopolymerization to producesize-controlled PEGDA hydrogel particles. The role of initial solutioncomposition on resultant particle size is reported, and is found tocontribute through its influence on the polymerization rate, as well asthe diffusivity of oxygen. By controlling photopolymerization kineticsfacilely via UV light intensity and/or exposure time, PEGDA particleswere produced with dimensions independent of the parent sphericaldroplets formed by conventional microfluidic emulsification.

Introduction

Biomaterials, such as polymers,¹ ceramics,² and metals³ are widely usedin biomedical diagnostic, therapeutic, and prosthetic applications.Among these, hydrogels, defined as water-swollen, cross-linkedhydrophilic polymer gels, have shown great potential for biological andmedical applications.⁴ Synthetic hydrogels have become a focus ofparticular interest in the last twenty years due to their well-definedstructure that can be modified to add functionality and programmeddegradability.⁵ In particular, hydrogels of poly(ethylene glycol)diacrylate (PEGDA) have been investigated for tissue engineering^(6,7)and drug delive⁸⁻¹¹ applications because of their biocompatibility,non-immunogenicity, resistance to protein adsorption, and adjustablemechanical properties and chemical composition.^(12,13) Functionalhydrogels can be tailored to possess well-defined permeability andstiffness,¹⁴ to be sensitive to temperature,¹⁵ and to degradehydrolytically,^(16,17) photolytically,^(18,19) or enzymatically.²⁰Among the advantages of PEGDA, its ability to be photopolymerized ismost notable, as it lends spatial and temporal control over hydrogelproperties,²¹ adding to its versatility and convenience.²²

Hydrogels are typically formulated as bulk structures, such as films andmonolithic molds, but emerging applications demand miniaturization fordelivery and transport in microscopic environments.²³ In comparison totraditional polymeric nanocarriers such as micelles, liposomes, andpolymerosomes, hydrogel particles in the micron and submicron size rangeoffer many advantages, such as controlled loading, versatility inmaterial composition and type of biological cargo, and physicalstability.²⁴ These particles have been previously synthesized by bulkemulsion and dispersion polymerization, which result in a highlypolydisperse particle size distribution.²⁵ More recently, dropletmicrofluidic particle templating has gained popularity due to itsaccurate control over particle size and dispersity.²⁸ Droplet-basedmicrofluidic systems utilize two flowing immiscible phases, usually incombination with a stabilizing surfactant, to form discrete droplets ata channel junction via interfacial instabilities.²⁷ The size of formeddroplets depends upon viscous forces, surface and interfacial chemistry,and channel geometry. However, while droplets in the 10-60 μm range havebeen reliably produced,²⁸ droplet production for <10 μm or evensubmicron is still challenging due to the high shear energy required toovercome the interfacial forces in aqueous solutions.

Microfluidic methods such as tipstreaming in a flow focusingdevice,^(29,30) electrospraying,³¹ satellite droplet collection,³² anddroplet shrinking^(33,34) have been used to obtain submicron droplets.However, these methods all possess shortcomings that constrain theirutility. Tipstreaming, for instance, requires a high viscosity ratiobetween the immiscible phases and high surfactant concentrations. As aresult, it is very sensitive to pressure fluctuations, and fails whenusing aqueous solutions with high macromer concentrations due toviscoelastic memory effects. Electrospraying requires high energy inputand very high flow rate ratios, and is dependent upon the conductivityof the liquids used. Moreover, none of these methods have been coupledwith in situ photopolymerization to continually produce hydrogelparticles, resulting in decreased monodispersity as a consequence ofrandom droplet coalescence during the collection process.

Photopolymerization of PEGDA droplets in microfluidic devices hasexperienced limited adoption due to challenges arising primarily fromoxygen inhibition effects. The inhibition of PEGDA photopolymerizationoccurs as a result of the rapid reaction of oxygen with photoinitiatorand propagating monomer radicals to form peroxides, resulting in no orincomplete polymerization where oxygen is present in surplus. ³⁵⁻³⁸ Allacrylates are inherently vulnerable to oxygen inhibition, and thus,dissolved oxygen must be almost completely consumed before thepolymerization reaction can occur. In the case of PEGDA, the consumptionof oxygen results in an induction period before which no PEG macromer isconverted to cross-linked hydrogel.³⁹ At oxygen rich interfaces, acompetition occurs between the photopolymerization reaction and oxygendiffusion due to the replenishment of oxygen. This is particularlyrelevant to the photopolymerization of thin films. ° and within gaspermeable polydimethylsiloxane (PDMS) microfluidic devices.^(41,42)Attempts to counteract the effects of oxygen inhibition include oxygenscavengers, reducing agents, potent photoinitiators, and purging with aninert gaS.^(43,44) It is possible to photopolymerize emulsion dropletsflowing within PDMS microfluidic channels by implementing one or more ofthese methods, but they either increase the complexity of themicrofluidic device design or require adding reactive chemical speciesinto the system, which is generally undesirable for biomaterialsapplications.

While oxygen inhibition is generally regarded as undesirable, stop flowlithography⁴⁵ and gradient-mediated photopatterning techniques⁴⁶ haveexploited the presence of oxygen in microfluidic devices to obtainuniquely shaped particles. In droplet microfluidics, the oxygeninhibition effect can be observed at the interface between aqueousdroplets and the surrounding oil. As reported by the co-author's group,this effect is pronounced when using oils with high oxygen solubilityand diffusivity, as they provide a constant flux of oxygen to thedroplets. Upon UV exposure, spherical hydrogel particles are polymerizedat the droplet center and are surrounded by an unpolymerized shell, thethickness of which has been shown to be independent of the total dropletdiameter.⁴⁷ This behavior, arising from equal rates of oxygen reactionand diffusion, presents a unique platform to obtain hydrogel particleswith smaller diameters than that of the droplet. FIG. 2 schematicallydescribes droplet photopolymerization in a microfluidic channel withoxygen present and illustrates how a single process parameter can bemanipulated to vary particle size within uniformly sized droplets. Inthis paper, we have quantified the effect of differentphotopolymerization parameters, including the UV dose and macromersolution composition, on the unpolymerized shell thickness. Thisplatform also allows the study of droplet photopolymerization kineticsby coupling empirical results with a reaction-diffusion model toquantify the sensitivity of each variable upon the photopolymerizationof hydrogel particles within microfluidic emulsion droplets.

Kinetic models for the photopolymerization of multifunctional acrylateshave been previously developed to accurately describe the effect ofoxygen inhibition on polymer coatings, membranes,^(48,49) and thinfilms.⁵⁰ These models have been adapted to microfluidic contexts todescribe and predict the size of a particle produced via stop flowlithography in microfluidic devices under different exposureconditions⁴⁵ and to explain the presence of an unpolymerized shellaround a hydrogel particle when photopolymerizing droplets in ambientconditions.⁴⁷ By understanding the influence of each parameter in thehydrogel photopolymerization process, we can exploit oxygen inhibitionto produce particles that are <10 μm or submicron from larger, easilyproduced droplet templates.

As described herein, we perform a systematic characterization of theeffects of hydrogel-forming parameters on oxygen-inhibited PEGDAphotopolymerization within emulsion droplets. The presentedreaction-diffusion model accurately captures stoichiometric andphotochemical effects on polymerization rate, and incorporates apreviously unappreciated relationship between solution viscosity andoxygen diffusivity. Having produced a reaction-diffusion model thatcompletely describes the full range of empirical results presented here,we have developed a quantitatively comprehensive and predictiveunderstanding of PEGDA photopolymerization within flowing emulsions.This result has broad implications for the continuous production ofbiocompatible hydrogel particles and the high-throughput encapsulationof biomolecules.

Experimental (Materials and Methods)

Hydrogel forming solutions: Solutions containing poly(ethylene glycol)diacrylate (PEGDA 700 and PEGDA 575, Aldrich Chemistry) in deionizedwater were prepared and used as the dispersed phase. Poly(ethyleneglycol) monoacrylate (PEGMA 400, PolySciences Inc.) and poly(ethyleneglycol) (PEG 400, Aldrich) were added to alter acrylate concentrationand solution viscosity. Monomethyl ether hydroquinone (MEHQ) was removedfrom PEGDA 575 with a prepacked adsorption column (Aldrich Chemistry).

Two initiators were tested separately:2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959,Aldrich Chemistry) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate(LAP), which was synthesized as previously described.⁵¹ Irgacure 2959has been a widely used photoinitiator due to its moderate watersolubility and biocompatibility, while LAP is quickly becoming a popularoption due to its enhanced absorption at higher wavelengths and highercytocompatibility.²² Initiator was incorporated from 0.1-0.5% (LAP) and2-4% (Irgacure 2959), as indicated.

Microfluidic device design and operation: A single layer, flow focusingmicrofluidic channel configuration was used to form droplets within acontinuous phase, Novec 7500 with 2% Picosurf surfactant (DolomiteMicrofluidics). Fluorocarbon oils have been used in dropletmicrofluidics because their low viscosity, high density, and highvolatility facilitate droplet production and recovery relative tomineral oil.⁵² Droplets were produced in a 50 μm deep channel, pinchingdroplets in a 40 μm wide and 250 μm long nozzle, and flowed downstreamto a 110 μm deep and 200 μm wide straight channel, in which theirvelocity was reduced to allow longer UV exposure times. Droplet diameterwas set at −100 μm by setting the dispersed phase flow rate to 5 μl/hr,while the continuous phase flow rate was set to 40 μl/hr and 30 μl/hr inthe droplet formation section and the downstream section, respectively.Two-layer microfluidic devices were fabricated using common softlithography techniques.⁵³ Briefly, a silicon wafer (Silicon Inc. USA)was first patterned with SU-8 3050 negative photoresist (MicroChem, MA,USA) at a thickness of 50 μm. Features were polymerized by collimated UVlight exposure (Omnicure S2000, USA) through a photomask (CAD/ArtServices, OR), and hard baked for 1 minute at 65° C. and 3 minutes at95° C. A second planar flow network was patterned with SU-8 3050 atthickness of 60 μm (for a total of 110 μm) by the same method, and aftera second hard baking for 1 minute at 65° C. and 4 minutes at 95° C., theuncured photoresist was removed using a developer (propylene glycolmonomethyl ether acetate, Sigma-Aldrich, USA).

Polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, MI) was pouredupon the silicon wafer and cured in a 70° C. oven for 4 hours. Theelastomer replica was removed, trimmed, and punched with a sharpened 20G dispensing needle (Brico Medical Supplies, Inc., USA) to fashion inletand outlet holes. To ensure a hydrophobic surface for aqueous dropletgeneration, Aquapel (PPG Industries) was injected into the device andflushed with nitrogen. Omitting this step allowed aqueous droplets toadhere to the channel walls, altering flow patterns within device. PEGDAsolutions and oil were loaded into disposable plastic syringes (1 ml,Becton Dickinson) and the flow of each component was independentlycontrolled by syringe pumps (Nemesys syringe pump, Cetoni, Germany).Fluidic inlets and outlet on the PDMS microfluidic device were connectedto fluid sources and collection reservoirs, respectively, via microboreTygon tubing (0.01″ ID×0.03″ OD, ND-100-80, United States Plastic Corp).

Photopolymerization: An inverted microscope (IX71, Olympus) and a20×objective (Olympus LUCPlanFLN 0.45Ph1) were used for imagingdroplets. An X-Cite 120LEDmini (Excelitas Technologies) fluorescentillumination system was used to provide white light, which was filteredthrough a DAPI filter cube for photopolymerization. Since this lightsource is not monochromatic, the intensity spectral output was measuredusing a LumaSpec 800 Optical Power Meter (Prior Scientific) (see FIG. 35). An LED light source was utilized for photopolymerization due to itsshort-term and long-term stability and easily varied intensity. Thetotal channel area exposed to UV light was determined by flooding thedevice with only the hydrogel forming macromere solution, assuring thatthe fluid was quiescent, and exposing the channel to UV using the20×objective. After 10 seconds of exposure, a lower magnificationobjective and a Brightfield filter cube were used to image thepolymerized structure. Using ImageJ, the diameter of the UV exposurearea was determined from the dimensions of the structure, and later usedin conjunction with the cross-sectional area and flow rate to determineexposure time, which under the previously indicated flow rate was 1.2 s.

Droplets traveling along the channel were polymerized by exposure to UVlight at discrete intensities. Images of exposed droplets were acquiredafter the droplets were collected in a downstream reservoir (FIG. 2A)and later processed using ImageJ to determine droplet size, particlesize, and unpolymerized shell thickness. The polymerized core of thedroplets has a significantly different refractive index (see FIG. 2C)than the macromer solution, enabling polymerized particles and totaldroplet diameters to be easily measured.

Viscosity and solubility measurements: Oxygen solubility was measuredusing an Orion Star A113 Dissolved Oxygen Benchtop Meter and a 083005MDOrion polarographic dissolved oxygen sensor. Viscosity was measuredusing a Brookfield LVDV-E Laboratory Viscometer and a UL closed tubeadapter.

Results and Discussion

A reaction-diffusion model has been developed to predict hydrogelstructure formation by quantitatively comparing PEGDAphotopolymerization kinetics and oxygen diffusion within dropletsflowing through PDMS microfluidic channels. This model was validated andoptimized through a series of systematic experiments in which processparameters were varied and the relative diameters of polymerizedparticles and the surrounding unpolymerized shell thickness weremeasured within a droplet of arbitrary diameter. The results presentedhere are reported as unpolymerized shell thickness as opposed toparticle size because it has been shown that the former is a directfunction of reaction kinetics and oxygen diffusion, whereas the latteris the geometric product of the unpolymerized shell thickness and thedroplet size. ⁴⁷ As such, using unpolymerized shell thickness allowsthese results to be extrapolated to photopolymerization within arbitrarysized droplets in order to obtain particles with specific sizes. Toaccurately relate polymerization kinetics to unpolymerized shellthickness, experimental measurements must be acquired under steady stateconditions. Steady state is achieved when the oxygen profile throughoutthe droplet no longer changes with time, indicating an equivalence ofoxygen consumption and diffusive flux at a fixed radial position.Additionally, an oxygen saturation boundary condition is assumed inorder to establish the oxygen flux across the droplet interface, fromthe oil phase to the aqueous phase. To confirm that steady state hadbeen reached in our experiments, and to verify the saturated oxygenboundary condition, a set of preliminary experiments were firstconducted.

The exposure time required to reach steady state was determined bytracking the photopolymerization of PEGDA and photoinitiator containingemulsion droplets in a quiescent reservoir over a period of severalseconds. The results, summarized in FIG. 3 , show that the exposure timerequired to reach steady state depends strongly upon UV intensity, andthat lower UV intensities yield larger unpolymerized shells and thussmaller particles from uniformly sized droplets. These results areconsistent with the hypothesis that the thickness of the unpolymerizedshell is proportional to the ratio of oxygen diffusion and consumptionrates. Above a minimum threshold exposure time visible hydrogelparticles appear and while their diameter does not change withincreasing exposure time, the degree of crosslinking does continue toincrease, as determined by the change in refractive index of thepolymerized gels. This exposure time, described previously as theinduction time, is the sum of the time required for the local oxygen tobe depleted and the time necessary for the oligomer to be cross-linkedinto a gel. For multifunctional oligomers, gelation occurs at about 1-2%double bond conversion.⁵⁴ This simplifies data collection, as there isno risk of underexposing and not reaching steady state particle size.

FIG. 3 also illustrates that UV intensity is a key variable that impactsphotopolymerization kinetics. Intensity can be easily changed witheither neutral-density filters or variable light attenuation controls inthe illumination system. To demonstrate the range of particle sizes thatcan be obtained for a given solution of constant composition, all theexperiments were conducted over a range of intensities from 0.19 to 1.77mW/cm². A logarithmic trend was observed in all experimental data sets,in which the unpolymerized shell thickness displayed minor variation athigher intensities, while increasing drastically at lower intensities.These results suggest that in order to photopolymerize small particlesfrom a larger droplet size, it is necessary to operate at relatively lowUV intensities. It is worth noting that induction times are longer atlower intensity, therefore requiring longer exposure times (FIG. 3A) anda highly stable and well-controlled UV source. Thus, to reliably producesmall particles, it is easier to polymerize smaller droplets at a higherintensity. Furthermore, FIG. 3B illustrates qualitatively that it ispossible to vary the degree of crosslinking by changing exposure time.⁵⁵The relative effects of UV exposure intensity and time are furtherdescribed herein.

Effect of spacing on oxygen boundary conditions: Oxygen concentrationboundary conditions at the droplet-oil interface were determined byexposing randomly positioned, variable spaced droplets in a microfluidicreservoir. As shown in FIG. 4A, there is no correlation between spacingand shell thickness, even when the droplets are in direct contact withother aqueous droplets. This indicates that no oxygen concentrationgradient exists in the oil surrounding the droplet; instead, the highoxygen diffusivity and solubility of the oil maintain a constant oxygenconcentration at the droplet boundary.

Effect of flow: Photopolymerization under flow was suspected to possiblyaffect the extent of droplet photopolymerization, as convective fluidmotion outside and within the droplet can enhance the transport ofchemical species. While external transport was shown to beinconsequential due to the high diffusivity of oxygen throughfluorocarbon oil, recirculating flows within the droplet couldpotentially enhance convective mixing and thus the transport of reactivespecies throughout the droplet. Significant convective mixing, inaddition to diffusive oxygen transport, would modify the resultingunpolymerized shell thickness. To establish whether the effect ofconvective transport must be considered, experiments were carried outusing the same droplet composition under laminar flow (Re=0.177) andunder quiescent conditions. The results show that there is no impact offlow on unpolymerized shell thickness (FIG. 4B) under the specified flowand exposure conditions. Therefore, for short exposure times (t<1.2 s),internal convection can be neglected within droplets flowing in linearchannels operating under laminar conditions. In the experimentsconducted, an exposure time of 1.2 seconds was generally sufficient toobserve particle gelation in the intensity range used forphotopolymerization (0.19 to 1.77 mW/cm²).

Effects of PEGDA concentration, chain length and molecular weight: Forbiomedical applications, the diffusion of different sized solutesthrough a hydrogel is a basic design criterion that can be tailored bychanging macromolecular architecture and crosslinking density.⁵⁷ Themesh size, tensile strength, and swelling ratio for a given PEGDAhydrogel particle can all be tuned by using PEGDA macromers with varyingmolecular weight and concentration in solution, and by controlling thedegree of crosslinking. ^(14,55) Therefore, it is important to determinethe impact of these variables on droplet photopolymerization kineticsand resulting particle size.

To determine the effect of acrylate functional group concentration onunpolymerized shell thickness, four different concentrations of PEGDA700, corresponding to 20, 30, 35, and 40 wt %, in combination with 17 mMLAP were exposed over a range of UV light intensity. The results (FIG. 5) show that higher concentrations of PEGDA 700 yield particles with asmaller unpolymerized shell thickness. We hypothesize that thiscorrelation is attributable to the increasing density of acrylatefunctional groups at higher PEGDA concentration, which accelerates thepropagation of polyacrylate chain growth and crosslinking. Fasterpolymerization kinetics push the polymerization boundary closer to theinterface, resulting in thinner steady state shell thicknesses.

The effect of PEGDA chain length on photopolymerization kinetics wasdetermined by replacing PEGDA 700 with a lower average molecular weightPEGDA molecule (575). Based solely upon photopolymerization kinetics(Equations 3-6), reaction rates are functions of the acrylate functionalgroup concentration, which react to generate propagating radicals. Chainlength is therefore not predicted to affect particle size if theconcentration of acrylate functional groups is held constant. To achieveconstant acrylate concentration, the PEGDA mass fraction was scaled inproportion to the length of the PEG chain. Four different concentrationsof PEGDA 575, corresponding to 15, 22.5, 30, and 35 wt %, in combinationwith 17 mM LAP were exposed to UV light over the same intensity range asthe PEGDA 700 solutions. The results (FIG. 6A) indicate that the samequalitative trend observed for PEGDA 700 holds true for PEGDA 575, whereincreasing concentrations of acrylate functional groups yieldprogressively smaller unpolymerized shell thicknesses. However, chainlength does empirically impact the absolute thickness of theunpolymerized shell, as similar acrylate concentrations of PEGDA 575yield thicker unpolymerized shell thickness (a.k.a., smaller particles)than their PEGDA 700 counterparts. This behavior has been previouslyobserved for thin films,^(58,59) in which a film thickness was reportedto be more closely correlated with PEGDA weight percent than acrylateconcentration, but has not been fully explained.

Effect of inhibitor present in PEGDA: Several parameters wereinvestigated in order to explain the observed dependence upon PEGDAmolecular weight. First, photopolymerization kinetics were characterizedin the presence and absence of inhibitors—other than oxygen—in theoligomer solution. Inhibitors are commonly added to commercial monomersto prevent premature polymerization during storage and shipment, butthey can either be removed prior to polymerization or used incombination of an excess of initiator to mitigate their effects.⁶⁰ Aspurchased, PEGDA 700 contains 100 ppm MEHQ/300 ppm BHT, while PEGDA 575contains 400-600 ppm MEHQ. To evaluate the impact of inhibitor presenceon the unpolymerized shell thickness, an inhibitor removal column wasused to remove the MEHQ from PEGDA 575, and the inhibitor-free macromersolution was polymerized under the same exposure conditions as themacromer solution with inhibitor. The results, summarized in FIG. 6B,show that there is no significant difference in unpolymerized shellthickness following inhibitor removal, except at very low UVintensities. Because these inhibitors use dissolved oxygen to stabilizepropagating radicals, their presence has little effect upon thephotopolymerization reaction, during which oxygen is quickly consumed byinitiator radicals prior to chain.^(61,62)

Effect of acrylate concentration: To further investigate the relativeeffects of PEGDA molecular weight and acrylate concentration, smallamounts of poly(ethylene glycol) monoacrylate (PEGMA 400) were used incombination with PEGDA 575. The addition of PEGMA provided a means toestablish constant acrylate concentration and solution viscosity, whilechanging the average macromer unit molecular weight. Further, theunreactive ends of PEGMA molecules modify hydrogel network properties,resulting in a decrease in the swelling ratio and an increase in themechanical modulus and crosslinking density.⁶³ Maintaining a constantacrylate concentration of ˜1 M, three different solutions containingdifferent ratios of PEGDA/PEGMA were prepared and exposed to UV underconstant initiator concentration. As shown in FIG. 6C, there is nosignificant difference in shell thickness at different PEGDA:PEGMAratios, which indicates that, when keeping acrylate concentration andsolution viscosity constant, the intrinsic distribution of PEG macromermolecular weight does not affect unpolymerized shell thickness byitself.

Oxygen solubility and diffusivity variation between PEGDA solutions:Changes to the concentration and molecular weight of PEGDA have beenshown to alter the oxygen solubility and viscosity of hydrogel-formingsolutions. ^(64,65) Oxygen solubility of the aqueous phase affects thesystem's initial and boundary conditions, as well as the oxygen fluxinto the droplet during photopolymerization. Previous work showed thatoxygen solubility is inversely proportional to PEG concentration andmolecular weight.⁶⁴ Moreover, viscosity has a significant impact onacrylate photopolymerization, due to its effect on the diffusivity ofreactive species within the system. Lower viscosity solutions favorsegmental mobility resulting in faster crosslinking rates, but this iscountered by an increase of oxygen diffusion into the droplet.⁶⁵

Oxygen solubilities for solutions with varying concentrations of PEGDA575 and 700 were measured and are reported in FIG. 35 . An overalldecrease in oxygen solubility is observed as PEGDA concentrationincreases, which partially explains the increase in shell thickness atlower concentrations, resulting from a higher rate of oxygen diffusioninto the droplet as a result of an increased oxygen concentrationdifference between the particle edge and the emulsion interface.However, the difference in oxygen solubility between solutions of thetwo different PEGDA molecular weights is not significant, Moreover,modeling results, which are discussed later, showed little sensitivityto these small changes in oxygen solubility, warranting the measurementof solution viscosity to explain the difference between these two datasets. As shown in FIG. 36 , there is a considerable viscosity differencebetween PEGDA 575 and 700 at similar acrylate concentrations. This isexpected, as a higher weight percent of PEGDA 700 is required to matchthe acrylate concentration in PEGDA 575 solutions. We hypothesized thatthis variation in viscosity when varying PEGDA molecular weight andconcentration has a larger effect on particle photopolymerization thanacrylate concentration. To further investigate the hypothesis thatsolution viscosity dramatically affects oxygen diffusivity, unreactivepoly(ethylene glycol) (PEG) was added to PEGDA solutions to increasesolution viscosity without affecting acrylate stoichiometry. Unreactivepoly(ethylene glycol) (PEG) has previously been used in combination withPEGDA to make porous hydrogels.^(66,67) The addition of 0.25 M PEG 400to a 0.45 M PEGDA 575 solution had a visible impact onphotopolymerization (FIG. 6D), which is attributable to an increase inviscosity from 4.07±0.2 cP to 6.29±0.2 cP. The resulting decrease in theunpolymerized shell thickness reaffirms the inverse relationship betweenviscosity and photopolymerization kinetics, which strongly affects thethickness of the unpolymerized zone. The observed behavior merits theinclusion of solution viscosity and its effect on oxygen diffusivity asa modeling parameter to obtain accurate predictions of the hydrogelformation process. Previously developed models adequately capturestoichiometric effects but have failed to incorporate this alteration inoxygen transport in solutions when varying PEGDA molecular weights andconcentrations, which can lead to large errors in the predictedbehavior.

Effect of photoinitiator concentration: We have shown that UV intensity,acrylate concentration, and viscosity-mediated oxygen solubility allaffect the competing rates of photopolymerization and oxygen diffusionthat determines the unpolymerized shell thickness. The selection ofphotoinitiator can also dramatically alter photopolymerization rateswhen all other parameters are fixed. Photoinitiators are generallyselected based on their absorption and efficiency, as well as theirsolubility and application compatibility. We used a lithiumacylphosphinate salt (lithium phenyl-2,4,6-trimethylbenzoylphosphinate(LAP)) with high water solubility and activity at UV wavelength ranges.Three discrete concentrations of LAP were used in combination with 0.5 MPEGDA 700. The results (FIG. 7 ) show that photoinitiator concentrationis inversely proportional to unpolymerized shell thickness. Higherphotoinitiator concentrations increase the concentration of radicalsavailable to consume oxygen, which pushes the steady state oxygenboundary closer to the aqueous-oil interface, resulting in a smallerunpolymerized shell.

Another photoinitiator,2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (12959),was used to compare initiator efficiency and absorption. I2959 has alsobeen used extensively in cell and biomolecule encapsulation applicationsdue to its relatively high water solubility, moderate efficiency atlower wavelengths, and overall biocompatibility. FIG. 8 compares theresults from the two photoinitiators, revealing stark kineticdifferences, requiring significantly different initiator concentrationsto obtain similar shell thicknesses.

An axisymmetric model to describe the effect of process parameters andphysical properties: We have developed an axisymmetric one-dimensional,steady-state model for free radical hydrogel photopolymerization underdifferent operating conditions. Specifically, we describe the spatialand temporal concentration of chemical species found within water-in-oilemulsion droplets in a microfluidic channel. With this model, we aim toquantify the effect of the operating parameters and variables on theunpolymerized shell thickness of a particle polymerized in situ. Theequations and parameters used, including kinetic and transportconstants, are included in Table 1.^(45,47,68) This model evaluates thespatial distribution of photopolymerization rates as a function ofchemical species concentration. When compared to the experimentalresults (FIG. 9 ), the predicted data shows close agreement, with thetrends established previously being confirmed by the model.

TABLE 1 Transport and kinetic parameters used in the reaction-diffusionmodel for the oxygen inhibited photopolymerization of PEGDA particleswithin droplets in a microfluidic device Parameter Description ValueUnit Source k_(p) Rate 2.165 × 10⁴ L/(mol s) 2 constant of poly-merization k_(t) Rate 2.516 × 10⁶ L/(mol s) 2 constant of termination bycoupling k_(O) ₂ Rate 3 ×10⁹ for LAP L/(mol s) 3, 4 constant of 4.2 ×10⁹ for I2959 3, 4 inhibition 5 × 10⁸ for 5, 6 by oxygen Acrylates k_(i)Rate 1.9 × 10⁸ for LAP L/(mol s) 4, 7 constant of 4.8 × 10⁵ for I2959 8,9 initiation φ Quantum 0.29 for I2959 10, 11 yield 0.35 for LAP 7 εExtinction FIG. 37 m²/mol 1 coefficient I Intensity Measured (FIG. 37)mW/cm² C_(O) ₂ _(—H) ₂ _(O) ^(sat) Saturation Measured (FIG. 35) mol/m³concen- tration of oxygen in water D_(M-H) ₂ _(O) Diffusivity 7.6 ×10⁻¹² m²/s Calculated of monomer (Eq. 7) in water* D_(R*-H) ₂ _(O)Diffusivity 1.1 × 10⁻¹¹ m²/s Calculated of initiator (Eq. 7) radical inwater* D_(M*-H) ₂ _(O) Diffusivity 0 m²/s 12 of prop- agating radical inwater D_(PI-H) ₂ _(O) Diffusivity 1.6 × 10⁻¹¹ m²/s Calculated ofinitiator (Eq. 7) in water*

The modeling results for acrylate concentration (FIGS. 9A-9B) werefitted by changing oxygen diffusivity, which is justified by theaforementioned change in viscosity under varying PEGDA concentration.Generalized equations for diffusion of small molecules in liquids, suchas the Wilke-Chang equation (Equation 8), show an inverseproportionality between diffusivity and solution viscosity. Whencomparing measured viscosity and the inverse of the fitted oxygendiffusivity for each PEGDA concentration (FIG. 36 ), there is a clearcorrelation between the two, validating the model's predictivecapability. While changes in oxygen diffusivity greatly affected thepredicted particle size, changes in oxygen solubility in the measuredrange (185-200 M) were shown to have a negligible impact on thepredicted oxygen concentration profile, and did not affect the predictedunpolymerized shell thickness at all (FIG. 37 ). Conversely, varyingdiffusivity values for all other mobile chemical species, includingphotoinitiator, monomer, and initiator radical, indicated no effect onthe predicted shell thickness. When viscous effects on oxygendiffusivity are ignored, the model shows that there is little effect onchanging acrylate concentration on the unpolymerized shell thickness.This matches previous reports³⁹ that have shown that the local decreasein oxygen concentration, which allows the polymerization reaction totake place, is independent of acrylate concentration.

FIG. 9C shows close agreement between collected data and modelpredictions when using LAP, without requiring a change in transportparameters, as the addition of small photoinitiator molecules does notimpact solution viscosity. Initially, the model predicted that nophotopolymerization would occur when using Irgacure2959 even at highconcentrations, due to its low molar extinction coefficient and pooroverlap with the operating spectral range (FIG. 38 ). Fitting k_(d)values resulted in a 30-fold difference between the predicted and fittedvalue (FIG. 9D), indicating a discrepancy between theoretical andexperimental kinetic behavior of this photoinitiator.

Oxygen-inhibited photopolymerization of PEGDA droplets in a microfluidicdevice was conducted under different operating conditions to obtainparticles over a wide range of sizes from parent droplets with a singlediameter. It has been shown that ratio of unpolymerized and polymerizeddroplet radius is governed by the relative rates of oxygen consumptionand diffusive replenishment. In turn, the steady state oxygen profile isinfluenced by a host of stoichiometric and physical properties inherentto the emulsion. Ultraviolet intensity, macromer concentration andmolecular weight, and initiator concentration and type wereexperimentally varied to quantify their individual effects on theunpolymerized shell surrounding the polymerized hydrogel particle.Preliminary experiments revealed that, beyond a critical induction time,hydrogel particle size remains unchanged despite a visible increase incrosslinking, presenting the intriguing possibility that hydrogelparticles with defined mechanical properties may be produced by simplymanipulating exposure time,⁵⁵ facilitating the production of tailoredhydrogel particles for drug delivery^(9,24) and tissue engineering.⁷

As hypothesized, UV intensity and particle size are directlyproportional, exhibiting increased sensitivity to intensity as particlesize decreased. Initiator stoichiometry also behaved in a predictablemanner, and revealed substantially different initiator activity betweenLAP and Irgacure 2959. Varying PEGDA concentration and molecular weightrevealed that acrylate stoichiometry has little effect on particle size;rather, physical changes in solution viscosity surpassed kineticeffects. The number of independent variables and their oftencounteracting effects on polymerization and diffusion demand thedevelopment of a robust predictive model. We have presented a refinedfinite element reaction-diffusion model that provides good agreementwith experimental results, indicating that we are able to preciselycontrol and quantitatively predict unpolymerized shell thickness toobtain specific particle sizes in droplets of arbitrary diameter. Byusing this technique, we have demonstrated that it is possible tocontinually produce monodisperse hydrogel particles in the micro- andnano- scale from larger, easily produced droplets, overcoming thedrawbacks of alternative microfluidic techniques that have limitedranges of application.^(29,30,32)

Supplemental Information

Model

Reactions

Where: PI: Photoinitiator, R*: Primary radical, M: Acrylate functionalgroup, M*: Propagating radical, O₂: Oxygen, P: Terminated polymer chain,ROO: Primary radical peroxide, MOO: Propagating radical peroxide

Kinetics

$\begin{matrix}{\frac{d\lbrack{PI}\rbrack}{dr} = {- {k_{d}\lbrack{PI}\rbrack}}} & \left\lbrack {{Eq}.\mspace{14mu} 1} \right\rbrack \\{k_{d} = {\left( \frac{\varphi}{N_{A}hc} \right){\int{{ɛ(\lambda)}{I(\lambda)}d\;\lambda}}}} & \left\lbrack {{Eq}.\mspace{14mu} 2} \right\rbrack \\{\frac{d\left\lbrack {R*} \right\rbrack}{dt} = {{2{k_{d}\left\lbrack {PI} \right\rbrack}} - {{k_{i}\left\lbrack {R*} \right\rbrack}\lbrack M\rbrack} - {{k_{t}\left\lbrack {R*} \right\rbrack}\left\lbrack {M*} \right\rbrack} - {{k_{O_{2}}\left\lbrack {R*} \right\rbrack}\left\lbrack O_{2} \right\rbrack}}} & \left\lbrack {{Eq}.\mspace{14mu} 3} \right\rbrack \\{\frac{d\lbrack M\rbrack}{dt} = {{- {{k_{i}\lbrack M\rbrack}\left\lbrack {R*} \right\rbrack}} - {{k_{p}\lbrack M\rbrack}\left\lbrack {M*} \right\rbrack}}} & \left\lbrack {{Eq}.\mspace{14mu} 4} \right\rbrack \\{\frac{d\left\lbrack {M*} \right\rbrack}{dt} = {{{k_{i}\left\lbrack {R*} \right\rbrack}\lbrack M\rbrack} - {{k_{t}\left\lbrack {M*} \right\rbrack}\left\lbrack {R*} \right\rbrack} - {k_{t}\left\lbrack {M*} \right\rbrack}^{2} - {{k_{O_{2}}\left\lbrack {M*} \right\rbrack}\left\lbrack O_{2} \right\rbrack}}} & \left\lbrack {{Eq}.\mspace{14mu} 5} \right\rbrack \\{\frac{d\left\lbrack O_{2} \right\rbrack}{dt} = {{- {{k_{O_{2}}\left\lbrack O_{2} \right\rbrack}\left\lbrack {M*} \right\rbrack}} - {{k_{O_{2}}\left\lbrack O_{2} \right\rbrack}\left\lbrack {R*} \right\rbrack}}} & \left\lbrack {{Eq}.\mspace{14mu} 6} \right\rbrack \\{p = {\frac{\lbrack M\rbrack}{\lbrack M\rbrack_{o}} - 1}} & \left\lbrack {{Eq}.\mspace{14mu} 7} \right\rbrack\end{matrix}$

Where: p: Extent of conversion

Mass transport

Wilke-Chang Equation

$\begin{matrix}{D_{i} = \frac{{7.4} \times 10^{- 8}T\sqrt{\alpha_{SV}M_{SV}}}{\eta_{SV}V_{b,a}^{0.6}}} & \left\lbrack {{Eq}.\mspace{14mu} 8} \right\rbrack\end{matrix}$

Where: D_(i): Diffusivity of small molecule in liquid phase system, T:Absolute temperature (K), α_(sv): Solvent association coefficient,M_(sv): Solvent molecular weight, V_(b): Molar volume at the normalboiling point of solute

$\begin{matrix}{{\frac{\partial c_{i}}{\partial t} + {\nabla{\cdot \left( {{- D_{i}}{\nabla c_{i}}} \right)}}} = R_{i}} & \left\lbrack {{Eq}.\mspace{14mu} 9} \right\rbrack\end{matrix}$

TABLE 2 Fitted oxygen diffusivity values for PEGDA solutions. (Eventhough diffusivity of mobile species was determined to vary depending onviscosity, a sensitivity analysis on the model showed little or noeffect of these diffusivity values on the model). PEGDA MolecularConcentration Oxygen Diffusivity Weight (M) (m²/s) 700 0.24 1.175 ×10⁻¹⁰ 0.38 7.172 × 10⁻¹¹ 0.5 2.900 × 10⁻¹¹ 575 0.21 2.027 × 10⁻¹⁰ 0.351.399 × 10⁻¹⁰ 0.45 9.020 × 10⁻¹¹

TABLE 3 Materials and Methods: Chemical species information. ChemicalName Abbreviation Vendor Catalog No. Poly(ethylene glycol) PEGDA 575Sigma-Aldrich 26570-48-9 diacrylate 575 Poly(ethylene glycol) PEGDA 700Sigma-Aldrich 26570-48-9 diacrylate 700 Poly(ethylene glycol) PEGMA 400Polysciences, Inc. 25736-86-1 monoacrylate 400 Poly(ethylene PEG 400Sigma-Aldrich 25322-68-3 glycol) 400 2-hydroxy-4′- Irgacure 2959Sigma-Aldrich 106797-53-9 (2-hydroxyethoxy)-2- methylpropiophenoneLithium phenyl-2,4,6- LAP Synthesized 85073-19-4 trimethyl- fromprotocol benzoylphosphinate 3-ethoxy- Novec 7500 Dolomite 297730-93-91,1,1,2,3,4,4,5,5,6,6,6- Microfluidics dodecafluoro-2-trifluoromethyl-hexane Picosurf (Poly- — Dolomite — fluorinatedMicrofluidics surfactant)

REFERENCES

-   (1) Griffith, L. G. Polymeric Biomaterials. Acta mater 2000, No. 48,    263-277.-   (2) Saenz, A.; Rivera-Muñoz, E.; Brostow, W.; Castaño, V. M. Ceramic    Biomaterials: an Introductory Overview; 1999; Vol. 21, pp 297-306.-   (3) Niinomi, M. Metallic Biomaterials. J Artif Organs 2008, 11 (3),    105-110.-   (4) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R.    Hydrogels in Biology and Medicine: From Molecular Principles to    Bionanotechnology. Adv. Mater. 2006, 18 (11), 1345-1360.-   (5) Ahmed, E. M. Hydrogel: Preparation, Characterization, and    Applications: a Review. Journal of Advanced Research 2015, 6 (2),    105-121.-   (6) Anseth, K. S.; Metters, A. T.; Bryant, S. J.; Martens, P. J.;    Elisseeff, J. H.; Bowman, C. N. In Situ Forming Degradable Networks    and Their Application in Tissue Engineering and Drug Delivery.    Journal of Controlled Release 2002, 78, 199-209.-   (7) Hoffman, A. S. Hydrogels for Biomedical Applications. Advanced    Drug Delivery Reviews 2012, 64, 18-23.-   (8) Datta, A. Characterization of Polyethylene Glycol Hydrogels for    Biomedical Applications. 2007, 1-116.-   (9) Hamidi, M.; Azadi, A.; Rafiei, P. Hydrogel Nanoparticles in Drug    Delivery. Advanced Drug Delivery Reviews 2008, 60 (15), 1638-1649.-   (10) Lin, C.-C.; Anseth, K. S. PEG Hydrogels for the Controlled    Release of Biomolecules in Regenerative Medicine. Pharm Res 2008, 26    (3), 631-643.-   (11) Oh, J. K.; Drumright, R.; Siegwart, D. J.; Matyjaszewski, K.    The Development of Microgels/Nanogels for Drug Delivery    Applications. Progress in Polymer Science 2008, 33 (4), 448-477.-   (12) Zhu, J. Bioactive Modification of Poly(Ethylene Glycol)    Hydrogels for Tissue Engineering. Biomaterials 2010, 31 (17),    4639-4656.-   (13) Turturro, M. V.; Rendón, D. M. V.; Tcymour, F.;    Papavasiliou, G. Kinetic Investigation of Poly(Ethylene Glycol)    Hydrogel Formation via Perfusion-Based Frontal Photopolymerization:    Influence of Free-Radical Polymerization Conditions on Frontal    Velocity and Swelling Gradients. Macromolecular Reaction Engineering    2013, 7 (2), 107-115.-   (14) Hagel, V.; Haraszti, T.; Boehm, H. Diffusion and Interaction in    PEG-DA Hydrogels. Biointerphases 2013, 8 (36), 1-9.-   (15) Ramanan, R. M. K.; Chellamuthu, P.; Tang, L.; Nguyen, K. T.    Development of a Temperature-Sensitive Composite Hydrogel for Drug    Delivery Applications. Biotechnol Progress 2006, 22 (1), 118-125.-   (16) Du, Y. J.; Lemstra, P. J.; Nijenhuis, A. J.; van Aert, H. A.    M.; Bastiaansen, C. ABA Type Copolymers of Lactide with    Poly(Ethylene Glycol). Kinetic, Mechanistic, and Model Studies.    Macromolecules 1995, 28, 2124-2132.-   (17) Harrane, A.; Leroy, A.; Nouailhas, H.; Garric, X.; Coudane, J.;    Nottelet, B. PLA-Based Biodegradable and Tunable Soft Elastomers for    Biomedical Applications. Biomed. Mater. 2011, 6 (6), 065006-065012.-   (18) Kharkar, P. M.; Kiick, K. L.; Kloxin, A. M. Design of Thiol-    and Light-Sensitive Degradable Hydrogels Using Michael-Type Addition    Reactions. Polym. Chem. 2015, 6 (31), 5565-5574.-   (19) Kloxin, A. M.; Tibbitt, M. W.; Anseth, K. S. Synthesis of    Photodegradablc Hydrogels as Dynamically Tunable Cell Culture    Platforms. Nature Protocols 2010, 5 (12), 1867-1887.-   (20) West, J. L.; Hubbell, J. A. Polymeric Biomaterials with    Degradation Sites for Proteases Involved in Cell Migration.    Macromolecules 1999, 32 (1), 241-244.-   (21) Yang, C.; DelRio, F. W.; Ma, H.; Killaars, A. R.; Basta, L. P.;    Kyburz, K. A.; Anseth, K. S. Spatially Patterned Matrix Elasticity    Directs Stem Cell Fate. Proc Natl Acad Sci USA 2016, 113 (31),    E4439-E4445.-   (22) Fairbanks, B. D.; Schwartz, M. P.; Bowman, C. N.; Anseth, K. S.    Photoinitiated Polymerization of PEG-Diacrylate with Lithium    Phenyl-2,4,6-Trimethylbenzoylphosphinate: Polymerization Rate and    Cytocompatibility. Biomaterials 2009, 30 (35), 6702-6707.-   (23) Helgeson, M. E.; Chapin, S. C.; Doyle, P. S. Hydrogel    Microparticics From Lithographic Processes: Novel Materials for    Fundamental and Applied Colloid Science. Current Opinion in Colloid    & Interface Science 2011, 16 (2), 106-117.-   (24) Buwalda, S. J.; Vermonden, T.; Hennink, W. E. Hydrogels for    Therapeutic Delivery: Current Developments and Future Directions.    Biomacromolecules 2017, 18 (2), 316-330.-   (25) Saunders, B.; Vincent, B. Microgel Particles as Model Colloids:    Theory, Properties and Applications. Advances in Colloid and    Interface Science 1999, No. 80, 1-25.-   (26) Zhu, P.; Wang, L. Passive and Active Droplet Generation with    Microfluidics: a Review. Lab Chip 2017, 17 (1), 34-75.-   (27) Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Droplet    Microfluidics. Lab Chip 2008, 8 (2), 198-23.-   (28) De Geest, B. G.; Urbanski, J. P.; Thorsen, T.; Demeester, J.;    Dc Smedt, S. C. Synthesis of Monodisperse Biodegradable Microgels in    Microfluidic Devices. Langmuir 2005, 21 (23), 10275-10279.-   (29) Anna, S. L.; Mayer, H. C. Microscale Tipstreaming in a    Microfluidic Flow Focusing Device. Phys. Fluids 2006, 18 (12),    121512-121514.-   (30) Jeong, W.-C.; Lim, J.-M.; Choi, J.-H.; Kim, J.-H.; Lee, Y.-J.;    Kim, S.-H.; Lee, G.; Kim, J.-D.; Yi, G.-R.; Yang, S.-M. Controlled    Generation of Submicron Emulsion Droplets via Highly Stable    Tip-Streaming Mode in Microfluidic Devices. Lab Chip 2012, 12 (8),    1446-1449.-   (31) Kim, H.; Luo, D.; Link, D.; Weitz, D. A.; Marquez, M.;    Cheng, Z. Controlled Production of Emulsion Drops Using an Electric    Field in a Flow-Focusing Microfluidic Device. Appl. Phys. Lett.    2007, 91 (13), 133106-3.-   (32) Tan, Y.-C.; Lee, A. P. Microfluidic Separation of Satellite    Droplets as the Basis of a Monodispersed Micron and Submicron    Emulsification System. Lab Chip 2005, 5 (10), 1178-6.-   (33) He, M.; Sun, C.; Chiu, D. T. Concentrating Solutes and    Nanoparticles Within Individual Aqueous Microdroplets. Anal. Chem.    2004, 76 (5), 1222-1227.-   (34) Sang, Y. Y. C.; Lorenceau, E.; Wahl, S.; Stoffel, M.;    Angelescu, D. E.; Höhler, R. A Microfluidic Technique for Generating    Monodisperse Submicron-Sized Drops. RSC Advances 2013, 3 (7),    2330-2336.-   (35) Gou, L.; Opheim, B.; Coretsopoulos, C. N.; Scranton, A. B.    Consumption of the Molecular Oxygen in Polymerization Systems Using    Photosensitized Oxidation of Dimethylanthracene. Chemical    Engineering Communications 2006, 193 (5), 620-627.-   (36) Hofer, M.; Moszner, N.; Liska, R. Oxygen Scavengers and    Sensitizers for Reduced Oxygen Inhibition in Radical    Photopolymerization. J. Polym. Sci. A Polym. Chem. 2008, 46 (20),    6916-6927.-   (37) Chong, J. Oxygen Consumption During Induction Period. Journal    of Applied Polymer Science 1969, 13, 241-247.-   (38) Ligon, S. C.; Husár, B.; Wutzel, H.; Holman, R.; Liska, R.    Strategies to Reduce Oxygen Inhibition in Photoinduced    Polymerization. Chem. Rev. 2014, 114 (1), 557-589.-   (39) Decker, C.; Jenkins, A. D. Kinetic Approach of O2 Inhibition in    Ultraviolet- and Laser-Induced Polymerizations. Macromolecules 1985,    1241-1244.-   (40) O'Brien, A. K.; Bowman, C. N. Impact of Oxygen on    Photopolymerization Kinetics and Polymer Structure. Macromolecules    2006, 39 (7), 2501-2506.-   (41) Merkel, T. C.; Bondar, V. I.; Nagai, K.; Freeman, B. D.;    Pinnau, I. Gas Sorption, Diffusion, and Permeation in    Poly(Dimethylsiloxane). Journal of Polymer Science Part B Polymer    Physics 2000, 38, 415-434.-   (42) McDonald, J. C.; Whitesides, G. M. Poly(Dimethylsiloxane) as a    Material for Fabricating Microfluidic Devices. Acc. Chem. Res. 2002,    35 (7), 491-499.-   (43) Miller, C. W.; Hoyle, C. E.; Jönsson, S.; Nason, C.; Lee, T.    Y.; Kuang, W. F.; Viswanathan, K. N-Vinylamides and Reduction of    Oxygen Inhibition in Photopolymerization of Simple Acrylate    Formulations. In Photoinitiated Polymerization; ACS Symposium    Series; American Chemical Society: Washington, D C, 2009; Vol. 847,    pp 2-14.-   (44) Kloosterboer, J. G.; Lijten, G. F. C. M.; Boots, H. M. J.    Network Formation by Chain Crosslinking Photopolymerization and Its    Applications in Electronics. Makromol. Chem. 1989, 24, 223-230.-   (45) Dendukuri, D.; Panda, P.; Haghgooie, R.; Kim, J. M.; Hatton, T.    A.; Doyle, P. S. Modeling of Oxygen-Inhibited Free Radical    Photopolymerization in a PDMS Microfluidic Device. Macromolecules    2008, 41 (22), 8547-8556.-   (46) Shim, T. S.; Yang, S.-M.; Kim, S.-H. Dynamic Designing of    Microstructures by Chemical Gradient-Mediated Growth. Nat Comms    2015, 6, 6584-6587.-   (47) Krutkramelis, K.; Xia, B.; Oakey, J. Monodisperse Polyethylene    Glycol Diacrylate Hydrogel Microsphere Formation by    Oxygen-Controlled Photopolymerization in a Microfluidic Device. Lab    Chip 2016, 16 (8), 1457-1465.-   (48) Kizilel, S.; Pérez-Luna, V. H.; Teymour, F. Mathematical Model    for Surface-Initiated Photopolymerization of Poly(Ethylene Glycol)    Diacrylate. Macromol. Theory Simul. 2006, 15 (9), 686-700.-   (49) Goodner, M. D.; Bowman, C. N. Development of a Comprehensive    Free Radical Photopolymerization Model Incorporating Heat and Mass    Transfer Effects in Thick Films. Chemical Engineering Science 2002,    57 (5), 887-900.-   (50) O'Brien, A. K.; Bowman, C. N. Modeling the Effect of Oxygen on    Photopolymerization Kinetics. Macromol. Theory Simul. 2006, 15 (2),    176-182.-   (51) Majima, T.; Schnabel, W.; Weber, W.    Phenyl-2,4,6-Trimethylbenzoylphosphinates as Water-Soluble    Photoinitiators. Generation and Reactivity of O=P(C6H5)(O—) Radical    Anions. Makromol. Chem. 1991, 2307-2315.-   (52) Holtze, C.; Rowat, A. C.; Agresti, J. J.; Hutchison, J. B.;    Angilè, F. E.; Schmitz, C. H. J.; Köster, S.; Duan, H.; Humphry, K.    J.; Scanga, R. A.; Johnson, J. S.; Pisignano, D.; Weitz, D. A.    Biocompatible Surfactants for Water-in-Fluorocarbon Emulsions. Lab    Chip 2008, 8 (10), 1632-1639.-   (53) Anderson, J. R.; Chiu, D. T.; Jackman, R. J.; Cherniavskaya,    O.; McDonald, J. C.; Wu, H.; Whitesides, S. H.; Whitesides, G. M.    Fabrication of Topologically Complex Three-Dimensional Microfluidic    Systems in PDMS by Rapid Prototyping. Anal. Chem. 2000, 72 (14),    3158-3164.-   (54) Andrzejewska, E. Photopolymerization Kinetics of    Multifunctional Monomers. Progress in Polymer Science 2001, 26 (4),    605-665.-   (55) Hwang, D. K.; Oakey, J.; Toner, M.; Arthur, J. A.; Anseth, K.    S.; Lee, S.; Zeiger, A.; Van Vliet, K. J.; Doyle, P. S. Stop-Flow    Lithography for the Production of Shape-Evolving Degradable Microgel    Particles. J. Am. Chem. Soc. 2009, 131 (12), 4499-4504.-   (56) Dendukuri, D.; Panda, P.; Haghgooie, R.; Kim, J. M.; Hatton, T.    A.; Doyle, P. S. Modeling of Oxygen-Inhibited Free Radical    Photopolymerization in a PDMS Microfluidic Device. Macromolecules    2008, 41 (22), 8547-8556.-   (57) Peppas, N.; Sahlin, J. A Simple Equation for the Description of    Solute Release. III. Coupling of Diffusion and Relaxation.    International Journal of Pharmaceutics 1989, No. 57, 169-172.-   (58) Cruise, G.; Scharp, D.; Hubbell, J. Characterization of    Permeability and Network Structure of Interfacially Photopolymerized    Poly(Ethylene Glycol) Diacrylate Hydrogels. Biomaterials 1998, No.    19, 1287-1294.-   (59) Kizilel, S.; Perez-Luna, V. H.; Teymour, F. Mathematical Model    for Surface-Initiated Photopolymerization of Poly(Ethylene Glycol)    Diacrylate. Macromol. Theory Simul. 2006, 15 (9), 686-700.-   (60) Odian, G. Principles of Polymerization; John Wiley & Sons,    Inc.: Hoboken, N.J., 2004; pp 1-839.-   (61) Burton, G. W.; Ingold, K. U. Autoxidation of Biological    Molecules. J. Am. Chem. Soc. 1981, No. 103, 6472-6477.-   (62) Becker, H.; Vogel, H. The Role of Hydroquinone Monomethyl Ether    in the Stabilization of Acrylic Acid. Chem. Eng. Technol. 2006, 29    (10), 1227-1231.-   (63) Beamish, J. A.; Zhu, J.; Kottke-Marchant, K.; Marchant, R. E.    The Effects of Monoacrylated Poly(Ethylene Glycol) on the Properties    of Poly(Ethylene Glycol) Diacrylate Hydrogels Used for Tissue    Engineering. J. Biomed. Mater. Res. 2009, 9999A, NA-NA.-   (64) Mexal, J.; Fisher, J.; Osteryoung, J.; Reid, C. P. P. Oxygen    Availability in Polyethylene Glycol Solutions and Its Implications    in Plant-Water Relations. Plant Physiology 1975, No. 55, 20-24.-   (65) Scherzer, T.; Langguth, H. Temperature Dependence of the Oxygen    Solubility in Acrylates and Its Effect on the Induction Period in UV    Photopolymerization. Macromol. Chem. Phys. 2005, 206 (2), 240-245.-   (66) Choi, N. W.; Kim, J.; Chapin, S. C.; Duong, T.; Donohue, E.;    Pandey, P.; Broom, W.; Hill, W. A.; Doyle, P. S. Multiplexed    Detection of mRNA Using Porosity-Tuned Hydrogel Microparticles.    Anal. Chem. 2012, 84 (21), 9370-9378.-   (67) Lee, A. G.; Arena, C. P.; Beebe, D. J.; Palecek, S. P.    Development of Macroporous Poly(Ethylene Glycol) Hydrogel Arrays    Within Microfluidic Channels. Biomacromolecules 2010, 11 (12),    3316-3324.-   (68) Xia, B.; Krutkramelis, K.; Oakey, J. Oxygen-Purged Microfluidic    Device to Enhance Cell Viability in Photopolymerized PEG Hydrogel    Microparticles. Biomacromolecules 2016, 17 (7), 2459-2465.

Supplemental

-   (1) Fairbanks, B. D.; Schwartz, M. P.; Bowman, C. N.; Anseth, K. S.    Photoinitiated Polymerization of PEG-Diacrylate with Lithium    Phenyl-2,4,6-Trimethylbenzoylphosphinate: Polymerization Rate and    Cytocompatibility. Biomaterials 2009, 30 (35), 6702-6707.-   (2) Kizilel, S.; Pérez-Luna, V. H.; Tcymour, F. Mathematical Model    for Surface-Initiated Photopolymerization of Poly(Ethylene Glycol)    Diacrylate. Macromol. Theory Simul. 2006, 15 (9), 686-700.-   (3) Jockusch, S.; Turro, N. J. Phosphinoyl Radicals: Structure and    Reactivity. a Laser Flash Photolysis and Time-Resolved ESR    Investigation. J Am. Chem. Soc. 1998, 120 (45), 11773-11777.-   (4) Colley, C. S.; Grills, D. C.; Besley, N. A.; Jockusch, S.;    Matousck, P.; Parker, A. W.; Towrie, M.; Turro, N. J.; Gill, P. M.    W.; George, M. W. Probing the Reactivity of Photoinitiators for Free    Radical Polymerization: Time-Resolved Infrared Spectroscopic Study    of Benzoyl Radicals. J. Am. Chem. Soc. 2002, 124 (50), 14952-14958.-   (5) Dendukuri, D.; Panda, P.; Haghgooie, R.; Kim, J. M.; Hatton, T.    A.; Doyle, P. S. Modeling of Oxygen-Inhibited Free Radical    Photopolymerization in a PDMS Microfluidic Device. Macromolecules    2008, 41 (22), 8547-8556.-   (6) Decker, C.; Jenkins, A. D. Kinetic Approach of O2 Inhibition in    Ultraviolet- and Laser-Induced Polymerizations. Macromolecules 1984,    18, 1241-1244.-   (7) Majima, T.; Schnabel, W.; Weber, W.    Phenyl-2,4,6-Trimethylbenzoylphosphinates as Water-Soluble    Photoinitiators. Generation and Reactivity of O=P(C6H5)(O—) Radical    Anions. Makromol. Chem. 1991, 2307-2315.-   (8) Hristova, D.; Gatlik, I.; Rist, G.; Dietliker, K.; Wolf, J.-P.;    Birbaum, J.-L.; Savitsky, A.; MÖbius, K.; Gescheidt, G. Addition of    Benzoyl Radicals to Butyl Acrylate: Absolute Rate Constants by    Time-Resolved EPR. Macromolecules 2005, 38 (18), 7714-7720.-   (9) Jockusch, S.; Turro, N. J. Radical Addition Rate Constants to    Acrylates and Oxygen: A-Hydroxy and A-Amino Radicals Produced by    Photolysis of Photoinitiators. J. Am. Chem. Soc. 1999, 121 (16),    3921-3925.-   (10) Scaiano, J. C.; Stamplecoskie, K. G.; Hallett-Tapley, G. L.    Photochemical Norrish Type I Reaction as a Tool for Metal    Nanoparticle Synthesis: Importance of Proton Coupled Electron    Transfer. Chem. Commun. 2012, 48 (40), 4798-11.-   (11) Jockusch, S.; Landis, M. S.; Freiermuth, B.; Turro, N. J.    Photochemistry and Photophysics of A-Hydroxy Ketones. Macromolecules    2001, 34 (6), 1619-1626.-   (12) Goodner, M. D.; Bowman, C. N. Development of a Comprehensive    Free Radical Photopolymerization Model Incorporating Heat and Mass    Transfer Effects in Thick Films. Chemical Engineering Science 2002,    57 (5), 887-900.

Example 2— Fabrication of Non-Spherical Hydrogel Particles for DrugDelivery Abstract

Hydrogel microparticles have become an intensively studied platform forthe delivery of drugs to treat many diseases, since they can be easilymodified to enhance treatment efficacy. Conventional particlefabrication methods usually generate spherical particles, but there isan interest in producing non-spherical particles because particle shapehas been shown to affect drug release profiles, particle degradation,transport in the body, and targeting abilities. There is a limitedavailability of techniques that produce non-spherical particles, amongstwhich microfluidics stands out because it allows precise dose control ofeach substance and facile functionalization of particle surfaces.However, current microfluidic methods are restricted by device geometryand capillary pressure, making it almost impossible to produce particlessmall enough (<10 μm) to be used for drug delivery applications. Usingpoly(ethylene glycol) diacrylate (PEGDA) as a model polymer platform, wedescribe a microfluidic technique to produce non-spherical hydrogelparticles by taking advantage of oxygen inhibition, often undesirable,to control droplet size and shape. Briefly, due to oxygen diffusion andinhibition of the radical photopolymerization reaction, dropletspolymerize from the core outward. This enables us to use larger andeasily produced micro-droplets to fabricate independently sizedparticles by controlling the outer unpolymerized shell thickness. In asimilar fashion, a variety of shapes, such as rods, discs, and wires,were produced by photopolymerizing deformed droplets as they traveleddown channels with varying dimensions. Using this method, we were ableto overcome the limitations in current microfluidic methods and producenon-spherical hydrogel.

Droplet Microfluidics

In droplet microfluidic, two immiscible phases interact to form discretedroplets due to high interfacial forces, a surfactant is used tostabilize these emulsions.¹ Microfluidic devices (as shown in FIG. 10 )offer precise control over droplet formation, and allow us to controlthe volume and concentrations within single droplets.² These devices areusually made from PDMS (Polydimethylsiloxane), a transparent and easilymolded material that isalso highly permeable to gases.³

Oxygen Inhibited Droplet Photopolymerization

Photoinitiated chain polymerization of acrylated PEG precursors can beused to form gels with specific properties. Advantages include highreaction rates and high spatial control. In microfluidic devices, oxygencan permeate through the PDMS and into the droplets, leading to oxygeninhibition, which reduces the rate of polymerization and forms peroxyradicals as described in FIG. 11 .^(4,5)

Size Control

Making homogeneous, monodisperse micro-scale droplets and polymerizingthem in ambient conditions allows us to fabricate particles independentof particle size as illustrated in FIG. 12 , reaching the lower limitsof particle size without as many device operation constraints.

Particle Size is proportional to: Droplet size, UV intensity, monomerconcentration, solution viscosity and initiator concentration.

Non-Spherical Particles

Shape, along with size and chemistry, is a critical feature of drugdelivery particles. Particle degradation and release profiles forencapsulated molecules will be dictated by particle shape. Transportalong the body will also be affected by particle shape due to particlevelocity, diffusion, and adhesion. The particle's targeting ability isalso dictated by the shape, since it impacts overall surface area andligand and opsonin adsorption, as well as fitting the contours of targetcell membranes.

Oblong and disk-shaped particles are illustrated in FIG. 13 .Disk-shaped, flexible particles with diameters of −10 μm routinely passthrough the spleen; spherical particles require diameters under 200 nmto accomplish the same task. Rod shaped and wire shaped particles areillustrated in FIG. 14 . Local curvature and surface area to volumeratio affect particle's targeting ability. More advanced shapes are alsodisclosed herein, for example, bullet shapes and particles aftermultiple exposures are provided in FIG. 15 . Multiple exposure particlescan possess regionally distinct mechanical properties.

Functional Surface

Surface chemistry influences the interactions of particles with cellsand tissues in the body. For drug delivery purposes, we can tailor thesurface properties to: 1) minimize recognition by the components of theimmune system and 2) target carriers to specific tissues using targetingligands such as antibodies and peptides.

Fully polymerized particles retain a surfactant layer on the surfacethat hinders functionality. We can wash the unpolymerized layer toobtain a surfactant-free functional surface, as described in FIG. 16 .

Acrylate-PEG-Biotin can be cross-linked into the hydrogel matrix asshown in FIG. 17 . Biotin groups will be present on the particlesurface, and in combination with a fluorescent avidin, displays visiblymeasurable activity. In FIG. 18 provides an example using 0.4%Ac-PEG-Biotin, 30% PEGDA 700 and 0.5% LAP. Fluorescent intensitycorrelates tobiotin groups that are present at the interface. Particlesexposed for less time show increased surface roughness. FIG. 19illustrates that surface activity decreases with increasing exposuretime due to crosslinking gradient at the particle surface.

REFERENCES

-   (1) Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Droplet    Microfluidics. Lab Chip 2008, 8 (2), 198-23.-   (2) Tan, Y.-C.; Fisher, J. S.; Lee, A. I.; Cristini, V.; Lee, A. P.    Design of Microfluidic Channel Geometries for the Control of Droplet    Volume, Chemical Concentration, and Sorting. Lab Chip-   (3) Kim, M.-C.; Lam, R. H. W.; Thorsen, T.; Asada, H. H.    Mathematical Analysis of Oxygen Transfer Through    Polydimethylsiloxane Membrane Between Double Layers of Cell Culture    Channel and Gas Chamber in Microfluidic Oxygenator. Microfluid    Nanofluid 2013, 15 (3), 285-296.-   (4) Dendukuri, D.; Panda, P.; Haghgooie, R.; Kim, J. M.; Hatton, T.    A.; Doyle, P. S. Modeling of Oxygen-Inhibited Free Radical    Photopolymerization in a PDMS Microfluidic Device. Macromolecules    2008, 41 (22), 8547-8556.-   (5) Krutkramelis, K.; Xia, B.; Oakey, J. Monodisperse Polyethylene    Glycol Diacrylate Hydrogel Microsphere Formation by    Oxygen-Controlled Photopolymerization in a Microfluidic Device. Lab    Chip 2016, 16 (8), 1457-1465.

Example 3—Engineering Functional Hydrogel Microparticle Interfaces byControlled Oxygen-Inhibited Photopolymerization Abstract

Functional poly(ethylene glycol) diacrylate (PEGDA) hydrogelmicroparticles for the detection of bioactive macromolecules werefabricated via oxygen-inhibited photopolymerization in a dropletmicrofluidic device. Hydrogel network functionalization and architecturewere characterized using a biotin-avidin binding assay, which revealedradial network inhomogeneities dependent on exposure conditions.Empirical results were corroborated using a reaction-diffusion model,describing the effects of exposure intensity on the spatialphotopolymerization kinetics and resulting polymeric mesh network. Thecombination of finely controlled exposure conditions and predictivesimulations enables the generation of tailored particles withmicroengineered interfaces and gradients in crosslinking density, whichdictate solute diffusivity and elasticity, augmenting the utility ofthis approach in engineering multifunctional, size-excluding hydrogelparticles for multiplexed biomolecular sensing.

Introduction

Synthetic, photopolymerized hydrogels are versatile and widely usedbiomaterials that consist of highly crosslinked, water-swollen,sample-spanning polymer networks. Techniques to tailor hydrogel networkproperties are commonly used to control the conjugation, stericencapsulation, and release of bioactive macromolecules. Hydrogelscomposed of poly(ethylene glycol) diacrylate (PEGDA) have attractedparticular and widespread interest due to their fabrication versatilityand high degree of tunability. Diacrylated PEG-containing blockcopolymers have been used to form hydrogels with dynamic networkproperties enabling them to be used for a diversity of applications invarious microenvironments. Photoinitiated polymerization lends exquisitespatial and temporal control over polymerization kinetics as well as theability to lithographically pattern PEGDA hydrogel structures withdimensions on the micrometer scale. Microscale hydrogels have been usedas drug delivery vehicles, tissue scaffolds, biosensors, and diagnosticplatforms.

Various applications for hydrogels benefit from the direct conjugationof functional moieties to the hydrogel network in order to impartheterogeneous spatial properties and function.^(10,11) Biosensingapplications particularly rely on the copolymerization of proteins andenzymes in the hydrogel network for detection and quantification ofspecific analytes.¹²⁻¹⁴ Hydrogel microparticle-based biosensingplatforms possess many advantages over benchtop techniques, such as lowcost, greater detection sensitivity, small sample consumption,multiplexing, and the ability to perform on-site analysis. ¹⁵⁻¹⁸Hydrogel microparticles have been produced via a variety of methods,including suspension, emulsion, and precipitation polymerization andphotolithographic techniques.¹⁹ Stop flow lithography (SFL) has manyadvantages over batch techniques due to its fine control over particlesize, shape, and network properties and has been used to fabricatebar-coded hydrogel microparticles for multiplexed protein and nucleicacid detection and quantification.^(20,21) The introduction of mesh sizegradients in hydrogel microparticles generated using SFL furtherincreases detection specificity by incorporating molecular sizeexclusion capabilities, enabling the size-selective sieving of moleculessuch as mRNA.^(22,23) Despite its versatility, SFL can only generateparticles with internal mesh size gradients via macromer molecularweight gradients formed by laminar flow lithography²¹ or gradientgenerators,²⁴ or through the use of porogens.²² SFL is also aninherently low throughput (<5 Hz) production method.²¹ To address thesechallenges, here we report a high-throughput technique that enables thecontinuous generation of hydrogel particles with complex and tunablenetwork properties without the need for porogens or graded materialprecursors

Droplet microfluidics, a technique used to produce emulsion droplets bymerging two coflowing immiscible fluid phases, has emerged as aversatile and high-throughput alternative, allowing the continuousproduction of monodisperse microparticles²⁵ that can be easilyfunctionalized.²⁵ Polydimethylsiloxane (PDMS) is commonly used toconstruct droplet microfluidic devices, because it is inexpensive,hydrophobic, and transparent, which facilitates imaging and coupling ofa UV light source to photopolymerize flowing droplets in situ.²⁷ Thehigh gas permeability of PDMS, combined with the high oxygen solubilityof fluorocarbon carrier oils commonly used for droplet formation,renders droplets polymerized in PDMS-based microchannels susceptible tooxygen inhibition.²⁸ This phenomenon refers to the quenching of primaryinitiating or propagating radicals by oxygen to form peroxyl radicals.Since peroxyl radicals do not initiate acrylate double bond conversion,radical chain polymerization cannot proceed until dissolved oxygen isconsumed almost completely.^(29,30) The competition between thediffusion and consumption of oxygen is evident in droplets due to theconstant replenishment of oxygen over the short diffusion length scalesacross the oil-aqueous interface to the aqueous core. As a consequence,exposed droplets possess a polymerized core and unpolymerized shell,with the thickness of shell being determined by the diffusion andreaction of oxygen. While generally deleterious to many biologicalapplications,³¹ oxygen inhibition has been previously exploited toproduce hydrogel particles in droplet templates with precise sizecontrol.³² Additionally, a predictive reaction-diffusion model wasdeveloped to fully describe particle-to-droplet diameter ratio as afunction of processing parameters during oxygen-inhibited particlephotopolymerization in emulsion droplets

Controlled oxygen-inhibited droplet photopolymerization presents aunique platform in which immunofunctional hydrogel particles withprecisely microengineered interfaces can be produced in ahigh-throughput fashion. Further, rigorous control over exposureconditions enables exquisite manipulation of the internal networkarchitecture of these particles without the need for porogens. Thistechnique is demonstrated by fabricating particles with biotin-decoratedhydrogel networks which are subsequently incubated within afluoro-neutravidin solution. Specific binding of the tagged ligandreveals the availability of copolymerized functional groups at theparticle surface and neutravidin's (NA) accessibility to the particleinterior. Additionally, experimental results are corroborated with areaction-diffusion model that describes the effect ofphotopolymerization kinetics on local hydrogel network architecture. Theintegrated approach of combining experimental results and modelprediction enables the fabrication of particles with a range ofcontrollable spatial gradients in their degree of crosslinking. Forexample, weakly crosslinked particles allow NA to diffuse across theirentire radius, while the highly crosslinked particles are completelyimpenetrable to NA. As crosslinking increases, biotin accessibilitybecomes sterically constrained entirely by the polymeric mesh network.These bounding cases highlight the tractability of this fabricationapproach and illustrate the importance of consideringphotopolymerization conditions when microfabricating particles forbiosensing applications. Particles fabricated by this platform can bedesigned with gradients in crosslinking density, diffusive conductivity,and elasticity, highlighting the utility of this approach in engineeringmultifunctional, size-excluding hydrogel particles for biomolecularsensing.

Experimental Section

Hydrogel forming solution preparation: A solution composed of 0.38 MPEGDA 700, 17 mM Lithium phenyl-2,4.6-trimethylbenzoylphosphinate (LAP),and 2.1 mM Acryl-PEG-Biotin (2000 Da) in a phosphate buffered saline(PBS, GenClone) was used as the aqueous phase, and a fluorocarbon oil(Novec 7500+2% Picosurf 1, Dolomite) was used as the oil phase for themicrofluidic droplet process.

Neutravidin-Rhodamine preparation: Neutravidin (Na) was conjugated toNHS-Rhodamine (NHS—Rh) according to the protocol provided by ThermoScientific, and diluted to a concentration of 0.43 mg/ml.

Microfluidic device preparation and operation: Flow focusing PDMSmicrofluidic devices were prepared using standard soft lithographytechniques. ³³ Briefly, a two layer photoresist pattern was fabricatedby two sequential UV exposure steps with two different photomasks toobtain different channel depths for each device section: 30 μm for thedroplet pinch-off section, and 110 μm for the downstreamphotopolymerization section.

Liquid flow was delivered to and controlled within the device using aseries of syringe pumps (neMESYS), flowing at 20 μl/hr for the aqueousphase, 40 μl/hr for the droplet pinch-off oil phase, and 140 μl/hr forthe downstream oil phase. At these flow rates, droplets with diametersof 100 μm were formed and exposed for 708±15 ms. Droplets werephotopolymerized using a white light LED source with a DAPI filter(350-400 nm) and a 10×objective (Olympus UPlanFLN 10×/0.30Ph1). Aftercollecting particles for 15 minutes, 1 ml of deionized water was added,and the mixture was vortexed for 30 s to wash off the unpolymerizedshell, after which the particles were separated from the oil and most ofthe water using a 5 μm centrifuge filter (1350 rpm for 5 min), andrecovered particles were resuspended in 400 μl of PBS. 30 μl aliquots ofthe particle mixture were combined with 7.5 μl of the Na—Rh solution andmixed for two days, after which the particles were washed andresuspended in 100 μl PBS, incubated for 2 days to allow unincorporatedNa—Rh to diffuse out, and imaged. For fluorescent imaging, a 20×objective was used in combination with a white light source filteredwith a Texas Red filter cube (Ex: 535-580 nm, Em: 590-670 nm). Ahigh-resolution camera (QIClick, QImaging) was used to collect images of30 particles for each exposure intensity, using an exposure time of 100ms. ImageJ was used to obtain an averaged radial profile of thefluorescent intensity for each particle, and after normalizing each dataset, the average profile was calculated.

Confocal images were acquired using an Olympus IX-81 microscope equippedwith a Yokogawa spinning disk (CSU X1) and a scientific complementarymetal-oxide semiconductor (sCMOS) camera (Orca-FLASH 4.0, Hammatsu. Twoobjectives (20× and 60×) were used to acquire images.

Results and Discussion

Photopolymerizable emulsions were formed using a flow-focusing PDMSmicrofluidic device and polymerized in situ. FIG. 39 is a schematicrepresentation of the copolymerization of acryl-PEG-biotin with PEGDA toform a hydrogel microparticle via photopolymerization in a microfluidicdevice under ambient conditions. Continuous generation of hydrogeldroplets within microfluidic devices enables facile and reproduciblecontrol over the composition of each droplet as it is being formed,presenting a unique advantage over batch polymerization processes. Uponexposure to UV light, a polymerized hydrogel core, surrounded by anunpolymerized shell is formed as a consequence of oxygen inhibition, aspreviously described.^(28,32) The thickness of this shell and,consequently, the size of the polymerized core can be easily tuned byadjusting the droplet composition and exposure intensity. Theunpolymerized shell, along with surfactant retained at the dropletinterface, can be removed via sonication and centrifugation to produceparticles with surface properties distinct from that of the parentdroplets. The ease of surfactant removal presents yet another advantageof oxygen-inhibited photopolymerization relative to fully-polymerizedparticles produced in an oxygen-depleted environment, in which thesurfactant molecules remain entangled in the hydrogel network at theinterface, requiring a second surfactant to re-suspend the particles inan aqueous solution. Surfactant-coated hydrogel interfaces can modifysurface interactions and even make the particle surface and functionalgroups tethered to it inaccessible.

To visualize and quantify the concentration and availability of biotingroups copolymerized in the hydrogel network during thephotopolymerization process, particles were incubated within a solutioncontaining Neutravidin-Rhodamine (NA-Rh), which diffused into thehydrogel and bound specifically to biotin. Subsequently hydrogelparticles were thoroughly washed and re-suspended in buffer. The strongavidin-biotin affinity retained NA-Rh within the particles, ensuringthat fluorescence observed after rinsing was the result of NA-Rh boundto the biotin in the hydrogel network. Fluorescent imaging of particles(FIG. 1B) revealed NA-Rh was distributed inhomogeneously throughout thehydrogel network, suggesting that either the hydrogel network or theavailability or activity of biotin displayed spatial variation. Imagingthe fluorescent intensity distribution within particles while stillimmersed in the NA-Rh solution show that the particle core isinaccessible to NA, even after long incubation times. After particlewashing, the gradient in fluorescent intensity became apparent,revealing that bound NA was retained in a radially varying manner. Ithas been previously reported that oxidized biotin does not lose affinityor binding capacity to avidin groups.³⁴ As Acryl-PEG-Biotin isincorporated into the hydrogel network in a statistical proportion toPEGDA,³⁵ it follows that the observed NA-Rh intensity traces thecrosslinking density of the hydrogel network.

To quantitatively explore this phenomenon, four discrete exposureintensities were used to photopolymerize droplets, while holding allother processing parameters, such as droplet composition and exposuretime, constant. Increasing exposure intensity accelerates the rate ofphotoinitiator radical production. In addition to reducing theunpolymerized shell thickness, increasing UV intensity should affect therate of macromer conversion and crosslinking. Particles would thereforebe generated with crosslinking maxima at the center, crosslinking minimaat their surface, and radial crosslinking density profiles that varynonlinearly with transient oxygen concentration duringphotopolymerization. As seen in FIG. 40B, particles produced withvarying exposure intensity indeed differ not only in size, but also inthe final distribution of NA-Rh throughout the particle. Clearly, theradial crosslinking density gradient within the hydrogel network allowedNA molecules, globular proteins with a molecular weight of roughly 60 kDa, to penetrate to a given radial position at which point the hydrogelmesh size becomes too constricted to allow NA diffusion. Thispenetration length can be visualized as a fluorescent shell surroundinga dark spherical core. This has been observed previously in hydrogelparticles for nucleic acid hybridization assays,¹⁶ but thephotopolymerization kinetics and their effect on hydrogel networkproperties were not described.

Radially-averaged NA-Rh fluorescent intensity profiles corresponding toeach UV exposure intensity used in FIG. 40 are summarized in FIG. 41 .Two distinct regions can be defined in the hydrogel network using theradial position of the maximum florescence intensity of this profile,r_(max) (FIG. 41 , indicated by a vertical dotted line). When r>r_(max),the fluorescent intensity corresponds to the availability of biotincopolymerized into the hydrogel network, and therefore can be used as adescriptor of the local crosslinking density. Since oxygen-inhibitedphotopolymerization within an emulsion droplet generates a radialconversion gradient, a biotin concentration gradient copolymerized withthe crosslinked network decays sharply near the particle surface. Forr<r_(max), the fluorescent intensity is no longer a function of thecopolymerized biotin concentration and decreases rapidly toward the morehomogeneously crosslinked core where very little or no detectablefluorescence can be observed. The exclusion of NA-Rh from the particlecore is a consequence of increased crosslinking density and thereforedecreased mesh size, which restricts the diffusion of NA-Rh.

To elucidate the relationship between oxygen inhibitedphotopolymerization kinetics and network architecture, a transientreaction-diffusion model was developed to fully describe hydrogelformation within a single droplet. A full description of this model canbe found in the Supplementary Information. The model enables predictionsof radial extent of conversion, the amount of macromer locallyincorporated into the hydrogel network, which is directly related to thedegree of crosslinking for bifunctional macromer units. The extent ofmacromer conversion, and thus crosslinking density, consequently dictatethe hydrogel's network properties. It has been previously shown that anincrease in the extent of conversion results in diminished permeabilityand diffusivity of solutes in PEGDA hydrogels.³⁶ FIG. 41 summarizes theobserved correlation between model prediction of extent of conversionand experimental intensity results. Both data sets are functions of thedroplet/particle radius. Previous reports suggest that gelation can beobserved above a minimum threshold conversion of 2%.³⁷ Previously, wevalidated the accuracy of the reaction-diffusion model in predictingparticle size from a particular droplet and given processingparameters.³² As shown in FIG. 41 , the particle size corresponds to thepredicted 2% extent of conversion value. It is instructive that for thefour different exposure intensity plots all four maxima linesintersected the respective predicted extent of conversion curves at thesame value of 38%. The consistent matching of the NA penetration cutoffto a predicted 38% extent of conversion confirms that the hydrogelnetwork surpasses a threshold crosslinking density at which NA-Rhdiffusion is restricted. This is further corroborated by FIG. 42 , whichsummarizes model predicted radial positions at which the extent ofconversion was 38% (dotted line). These predictions accurately describeempirical observations for the observed NA penetration depth.

Developing an understanding of the coupled oxygen and macromerconversion gradients is important in the design of hydrogels forapplications other than biosensing, such as drug delivery, in whichwater content, gel swelling, hydrolytic degradation rate, modulus,stiffness, and hydrophobicity are all affected by the crosslinkingdensity.³⁸ Additionally, fine-tuned control of oxygen-inhibitedphotopolymerization presents a unique opportunity to optimize theavailability of functional groups within and at the surface of hydrogelparticles. Beyond a threshold conversion, copolymerized functionalgroups will strictly be accessible at the interface, since the hydrogelnetwork mesh will be too highly crosslinked to allow molecular diffusioninto the network. Since the threshold conversion scales inversely withsolute size, carefully decreasing the conversion will enable diffusioninto the network with well-controlled penetration lengths. Forbiosensing applications, dense networks that are highly crosslinked atthe particle surface suppress sensitivity by presenting fewer accessiblefunctional groups. In contrast, lower UV intensity produced open networkarchitectures with well-defined gradients, which are useful forenhancing bioassay detection sensitivity of biomolecules with differentsizes.²²

Other processing variables, such as exposure time, monomerconcentration, and monomer chain length, will also impact the gradientformation process and final network architecture of hydrogelmicroparticles in a predictable manner. Well-known models are able topredict solute diffusion based solely on solution stoichiometry butdisregard extent of conversion effects.³⁹ Likewise, PEGDA hydrogelnetwork architecture and its dependent properties are typically reportedin terms of monomer concentration and molecular weight, disregarding theeffects of exposure conditions.^(40,41) A comprehensive model thatincorporates these effects will be instructive in designing particleswith specific network properties for a diverse number of applications,including tuning particle degradation rates, release profiles ofencapsulated components, and interaction with microenvironments bymodulating particle elasticity. The careful control of hydrogelcrosslinking density gradients can also be exploited to generate uniquemechanical properties for tissue engineering. While results here werecollected using particles with a size range of 60-100 μm, they may beextrapolated to larger or smaller particles produced usingoxygen-inhibited photopolymerization.

CONCLUSION

Described herein is a hydrogel microparticle fabrication platform thatenables the generation of radial crosslinking density gradients byexploiting oxygen-inhibited photopolymerization in a microfluidicdevice. This platform overcomes the limitations of previously reportedtechniques, such as SFL, by facilitating the continuous, high-throughputgeneration of custom hydrogel particles without the need for porogens orgraded material precursors.^([21-23]) The application of these particlesas macromolecular biosensors by incorporating biofunctional moleculeswas demonstrated using a model biotin-avidin assay, which revealednetwork architecture dependence on local gelation kinetics and operatingparameters. The generation of radial crosslinking density gradients wasempirically observed as a diffusive limitation of NA-Rh into theparticle past a critical penetration depth, which was dependent on UVexposure intensity. Experimental observations were corroborated with areaction-diffusion model that predicted a constant threshold conversionof 38%, which matched the penetration depth for different exposureintensities. The accurate predictive capabilities provided by thisreaction-diffusion model can be applied to easily design hydrogelparticles with custom network architectures based on photopolymerizationconditions. While this work focused on the detection of mobile speciesfollowing their diffusion into the particle, these particles can also beutilized in drug delivery and tissue engineering applications, where thecareful modulation of particle degradation and molecular release iscritical.

REFERENCES

-   (1) Liu, A. L.; Garcia, A. J. Methods for Generating Hydrogel    Particles for Protein Delivery. Annals of Biomedical Engineering    2016, 44 (6), 1946-1958.-   (2) Hwang, D. K.; Oakey, J.; Toner, M.; Arthur, J. A.; Anseth, K.    S.; Lee, S.; Zeiger, A.; Van Vliet, K. J.; Doyle, P. S. Stop-Flow    Lithography for the Production of Shape-Evolving Degradable Microgel    Particles. J. Am. Chem. Soc. 2009, 131 (12), 4499-4504.-   (3) DeForest, C. A.; Polizzotti, B. D.; Anseth, K. S. Sequential    Click Reactions for Synthesizing and Patterning Three-Dimensional    Cell Microenvironments. Nature Materials 2009, 8 (8), 659-664.-   (4) Kloxin, A. M.; Tibbitt, M. W.; Anseth, K. S. Synthesis of    Photodegradable Hydrogels as Dynamically Tunable Cell Culture    Platforms. Nature Protocols 2010, 5 (12), 1867-1887.-   (5) Zhu, J. Bioactive Modification of Poly(Ethylene Glycol)    Hydrogels for Tissue Engineering. Biomaterials 2010, 31 (17),    4639-4656.-   (6) Ahmad, M.; Rai, S. M.; Mahmood, A. Hydrogel Microparticles as an    Emerging Tool in Pharmaceutical Field: a Review. Adv. Polym.    Technol. 2015, 35 (2), 121-128.-   (7) Burdick, J. A.; Anseth, K. S. Photoencapsulation of Osteoblasts    in Injectable RGD-Modified PEG Hydrogels for Bone Tissue    Engineering. Biomaterials 2002, No. 23, 4315-4323.-   (8) Buenger, D.; Topuz, F.; Groll, J. Hydrogels in Sensing    Applications. Progress in Polymer Science 2012, 37 (12), 1678-1719.-   (9) Lee, A. G.; Arena, C. P.; Beebe, D. J.; Palecek, S. P.    Development of Macroporous Poly(Ethylene Glycol) Hydrogel Arrays    Within Microfluidic Channels. Biomacromolecules 2010, 11 (12),    3316-3324.-   (10) Sakhalkar, H. S. Enhanced Adhesion of Ligand-Conjugated    Biodegradable Particles to Colitic Venules. The FASEB Journal 2005,    1-19.-   (11) Burdick, J. A.; Khademhosseini, A.; Langer, R. Fabrication of    Gradient Hydrogels Using a Microfluidics/Photopolymerization    Process. Langmuir 2004, 20 (13), 5153-5156.-   (12) Yadavalli, V. K.; Koh, W.-G.; Lazur, G. J.; Pishko, M. V.    Microfabricated Protein-Containing Poly(Ethylene Glycol) Hydrogel    Arrays for Biosensing. Sensors and Actuators B: Chemical 2004, 97    (2-3), 290-297.-   (13) Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hammond, P. W.;    Kenney, M.; Boles, T. C. Immobilization of Acrylamide-Modified    Oligonucleotides by Co-Polymerization. Nucleic Acids Res 1999, 27    (2), 649-655.-   (14) Kenney, M.; Ray, S.; Boles, T. C. Mutation Typing Using    Electrophoresis and Gel-Immobilized Acrydite. BioTechniques 1998, 25    (3), 516-521.-   (15) Lee, Y.; Choi, D.; Koh, W.-G.; Kim, B. Poly(Ethylene Glycol)    Hydrogel Microparticles Containing Enzyme-Fluorophore Conjugates for    the Detection of Organophosphorus Compounds. Sensors and Actuators    B: Chemical 2009, 137 (1), 209-214.-   (16) Lewis, C. L.; Choi, C.-H.; Lin, Y.; Lee, C.-S.; Yi, H.    Fabrication of Uniform DNA-Conjugated Hydrogel Microparticles via    Replica Molding for Facile Nucleic Acid Hybridization Assays. Anal.    Chem. 2010, 82 (13), 5851-5858.-   (17) Le Goff, G. C.; Srinivas, R. L.; Hill, W. A.; Doyle, P. S.    Hydrogel Microparticles for Biosensing. European Polymer Journal    2015, 72 (C), 386-412.-   (18) Pregibon, D.; Toner, M.; Doyle, P. Multifunctional Encoded    Particles for High-Throughput Biomolecule Analysis. Science 2007,    315, 1393-1396.-   (19) Helgeson, M. E.; Chapin, S. C.; Doyle, P. S. Hydrogel    Microparticles From Lithographic Processes: Novel Materials for    Fundamental and Applied Colloid Science. Current Opinion in Colloid    & Interface Science 2011, 16 (2), 106-117.-   (20) Appleyard, D. C.; Chapin, S. C.; Srinivas, R. L.; Doyle, P. S.    Bar-Coded Hydrogel Microparticles for Protein Detection: Synthesis,    Assay and Scanning. Nature Protocols 2011, 6 (11), 1761-1774.-   (21) Bong, K. W.; Chapin, S. C.; Doyle, P. S. Magnetic Barcoded    Hydrogel Microparticles for Multiplexed Detection. Langmuir 2010, 26    (11), 8008-8014.-   (22) Choi, N. W.; Kim, J.; Chapin, S. C.; Duong, T.; Donohue, E.;    Pandey, P.; Broom, W.; Hill, W. A.; Doyle, P. S. Multiplexed    Detection of mRNA Using Porosity-Tuned Hydrogel Microparticles.    Anal. Chem. 2012, 84 (21), 9370-9378.-   (23) Luchini, A.; Geho, D. H.; Bishop, B.; Tran, D.; Xia, C.;    Dufour, R. L.; Jones, C. D.; Espina, V.; Patanarut, A.; Zhou, W.;    Ross, M. M.; Tessitore, A.; Petricoin, E. F.; Liotta, L. A. Smart    Hydrogel Particles: Biomarker Harvesting: One-Step Affinity    Purification, Size Exclusion, and Protection Against Degradation.    Nano Lett. 2008, 8 (1), 350-361.-   (24) Mahadik, B. P.; Wheeler, T. D.; Sketch, L. J.; Kenis, P. J. A.;    Harley, B. A. C. Microfluidic Generation of Gradient Hydrogels to    Modulate Hematopoietic Stem Cell Culture Environment. Adv.    Healthcare Mater. 2013, 3 (3), 449-458.-   (25) Teh, S.-Y.; Lin, R.; Hung, L.-H.; Lee, A. P. Droplet    Microfluidics. Lab Chip 2008, 8 (2), 198-23.-   (26) Kim, J. H.; Jeon, T. Y.; Choi, T. M.; Shim, T. S.; Kim, S.-H.;    Yang, S.-M. Droplet Microfluidics for Producing Functional    Microparticles. Langmuir 2014, 30 (6), 1473-1488.-   (27) Dang, T.-D.; Kim, Y. H.; Kim, H. G.; Kim, G. M. Preparation of    Monodisperse PEG Hydrogel Microparticles Using a Microfluidic    Flow-Focusing Device. Journal of Industrial and Engineering    Chemistry 2012, 18 (4), 1308-1313.-   (28) Krutkramelis, K.; Xia, B.; Oakey, J. Monodisperse Polyethylene    Glycol Diacrylate Hydrogel Microsphere Formation by    Oxygen-Controlled Photopolymerization in a Microfluidic Device. Lab    Chip 2016, 16 (8), 1457-1465.-   (29) Decker, C.; Jenkins, A. D. Kinetic Approach of O2 Inhibition in    Ultraviolet- and Laser-Induced Polymerizations. Macromolecules 1985,    1241-1244.-   (30) Ligon, S. C.; Husár, B.; Wutzel, H.; Holman, R.; Liska, R.    Strategies to Reduce Oxygen Inhibition in Photoinduced    Polymerization. Chem. Rev. 2014, 114 (1), 557-589.-   (31) Xia, B.; Jiang, Z.; Debroy, D.; Li, D.; Oakey, J.    Cytocompatible Cell Encapsulation via Hydrogel Photopolymerization    in Microfluidic Emulsion Droplets. Biomicrofluidics 2017, 11 (4),    044102-044111.-   (32) Debroy, D.; Oakey, J.; Li, D. Interfacially-Mediated Oxygen    Inhibition for Precise and Continuous Poly(Ethylene Glycol)    Diacrylate (PEGDA) Particle Fabrication. Journal of Colloid And    Interface Science 2018, 510, 334-344.-   (33) Xia, Y.; Whitesides, G. M. Soft Lithography. Annu. Rev. Mater.    Sci. 1998, 153-185.-   (34) Melville, D.; Genghof, D.; Lee, J. Biological Properties of    Biotin D- and L- Sulfoxides. J. Biol. Chem. 1954, No. 208, 503-512.-   (35) Beamish, J. A.; Zhu, J.; Kottke-Marchant, K.; Marchant, R. E.    The Effects of Monoacrylated Poly(Ethylene Glycol) on the Properties    of Poly(Ethylene Glycol) Diacrylate Hydrogels Used for Tissue    Engineering. J. Biomed. Mater. Res. 2009, 9999A, NA-NA.-   (36) Anseth, K. S.; Metters, A. T.; Bryant, S. J.; Martens, P. J.;    Elisseeff, J. H.; Bowman, C. N. In Situ Forming Degradable Networks    and Their Application in Tissue Engineering and Drug Delivery.    Journal of Controlled Release 2002, 78, 199-209.-   (37) Andrzejewska, E. Photopolymerization Kinetics of    Multifunctional Monomers. Progress in Polymer Science 2001, 26 (4),    605-665.-   (38) Lin, C.-C.; Anseth, K. S. PEG Hydrogels for the Controlled    Release of Biomolecules in Regenerative Medicine. Pharm Res 2008, 26    (3), 631-643.-   (39) Lustig, S.; Peppas, N. A. Solute Diffusion in Swollen    Membranes. IX. Scaling Laws for Solute Diffusion in Gels. Journal of    Applied Polymer Science 1988, 735-747.-   (40) Hagel, V.; Haraszti, T.; Boehm, H. Diffusion and Interaction in    PEG-DA Hydrogels. Biointerphases 2013, 8 (36), 1-9.-   (41) Cruise, G.; Scharp, D.; Hubbell, J. Characterization of    Permeability and Network Structure of Interfacially Photopolymerized    Poly(Ethylene Glycol) Diacrylate Hydrogels. Biomaterials 1998, No.    19, 1287-1294.

Statements Regarding Incorporation by Reference and Variations

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers of the group members, are disclosed separately. When a Markushgroup or other grouping is used herein, all individual members of thegroup and all combinations and subcombinations possible of the group areintended to be individually included in the disclosure. When a compoundis described herein such that a particular isomer, enantiomer ordiastereomer of the compound is not specified, for example, in a formulaor in a chemical name, that description is intended to include eachisomer of the compound described individual or in any combination.Additionally, unless otherwise specified, all isotopic variants ofcompounds disclosed herein are intended to be encompassed by thedisclosure. For example, it will be understood that any one or morehydrogens in a molecule disclosed can be replaced with deuterium ortritium. Isotopic variants of a molecule are generally useful asstandards in assays for the molecule and in chemical and biologicalresearch related to the molecule or its use. Methods for making suchisotopic variants are known in the art. Specific names of compounds areintended to be exemplary, as it is known that one of ordinary skill inthe art can name the same compounds differently.

Many of the molecules disclosed herein contain one or more ionizablegroups [groups from which a proton can be removed (e.g., —COOH) or added(e.g., amines) or which can be quaternized (e.g., amines)]. All possibleionic forms of such molecules and salts thereof are intended to beincluded individually in the disclosure herein. With regard to salts ofthe compounds herein, one of ordinary skill in the art can select fromamong a wide variety of available counterions those that are appropriatefor preparation of salts of this invention for a given application. Inspecific applications, the selection of a given anion or cation forpreparation of a salt may result in increased or decreased solubility ofthat salt.

Every formulation or combination of components described or exemplifiedherein can be used to practice the invention, unless otherwise stated.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

We claim:
 1. A method of generating a plurality of microparticles comprising: providing a continuous phase comprising a non-aqueous liquid and a dispersed phase comprising an aqueous solution including a functionalized monomer or a macromer and a photoinitiator; forming a composition comprising microdroplets of said aqueous solution dispersed in said non-aqueous liquid via a surfactant; diffusing oxygen through said non-aqueous liquid into said microdroplets; partially polymerizing a first microdroplet, thereby generating a first microparticle covered in an unpolymerized layer of aqueous solution, the first microparticle being smaller than the first microdroplet; and removing the unpolymerized layer of aqueous solution to expose a surfactant-free functionalized surface; wherein the functionalized monomer or macromer and the functionalized surface comprise functional groups configured for selective molecular binding.
 2. The method of claim 1, wherein the step of diffusing oxygen into said microdroplets generates an oxygen concentration gradient in the first microdroplet.
 3. The method of claim 2, wherein said oxygen concentration gradient results in a crosslinking gradient in the first microparticle.
 4. The method of claim 3, comprising controlling the crosslinking gradient via oxygen concentration during the partially polymerizing step such that molecules larger than a predetermined size are prevented from diffusing into the microparticle.
 5. The method of claim 1, wherein said step of partially polymerizing the first microdroplet comprises inhibiting polymerization via oxygen.
 6. The method of claim 1, wherein said step of partially polymerizing the first microdroplet comprises exposing the first microdroplet to UV light.
 7. The method of claim 1, wherein the composition is formed in a microfluidic device, the method comprising flowing the microdroplets through one or more channels of the microfluidic device.
 8. The method of claim 1, wherein said microdroplets have an average primary cross-sectional dimension of less than or equal to 100 μm and wherein the microparticle has a primary cross-sectional dimension of less than or equal to 20 μm.
 9. The method of claim 1 comprising treating the functionalized surface of said microparticle with a biological material.
 10. The method of claim 1, wherein said aqueous phase further comprises a biological material.
 11. A microparticle generated using the method of claim
 1. 12. The method of claim 1, wherein the aqueous solution includes the functionalized monomer or a macromer and a crosslinking hydrogel backbone monomer or macromer.
 13. The method of claim 12, wherein the partially polymerizing step comprises forming a crosslinked copolymer with functional groups of the crosslinked copolymer exposed to an exterior of the microparticle.
 14. The method of claim 1, wherein the functionalized surface of the microparticle is configured to exhibit at least one of: selective binding of particular species, biological functions, pharmacological functions and combinations thereof.
 15. The method of claim 1, wherein the partially polymerizing step comprises controlling the extent of conversion to below a threshold value in order to allow a target molecule to diffuse into a core of the microparticle.
 16. The method of claim 15, wherein the threshold value is selected based on a size of the target molecule.
 17. The method of claim 16 wherein the threshold value is less than 50%.
 18. The method of claim 16 wherein the threshold value is 38%.
 19. The method of claim 1, wherein the functional groups are configured for the detection of bioactive macromolecules.
 20. The method of claim 1, wherein the functional groups are configured for molecular recognition.
 21. The method of claim 20, wherein the molecular recognition comprises ligand-protein binding.
 22. The method of claim 1, wherein the functional groups comprise ligands. 